Secondary literature sources for Aspzincin_M35

The following references were automatically generated.

Takahashi S, Yoshiya T, Yoshizawa-Kumagaye K, Sugiyama T
Nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in soybean.
Biomed Res. 2015; 36: 219-24
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Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher N(epsilon)-2,4- dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates "Nma-Xaa-Pro-Lys(Dnp)" were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The Km, kcat, and kcat/Km values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 muM, 167 s(-1), and 7.17 muM(-1) s(-1), respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicotianamine. ACE2iSB strongly inhibited rhACE2 activity with an IC50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.

Baba R, Hori Y, Kikuchi K
Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity.
Chemistry. 2015; 21: 4695-702
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Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

Hardes K, Zouhir Hammamy M, Steinmetzer T
Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A.
Anal Biochem. 2013; 442: 223-30
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Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73-80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH2, a moderate kcat/Km value of 34s(-1)M(-1) was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A2( *), which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, kcat/Km values of 8010 and 8660s(-)(1)M(-)(1), respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH2)4-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH2 (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A2( *) activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH2, which was found to be a potent irreversible inhibitor of FXIII-A2( *).

Takahashi S, Ono H, Gotoh T, Yoshizawa-Kumagaye K, Sugiyama T
Novel internally quenched fluorogenic substrates for angiotensin I-converting enzyme and carboxypeptidase Y.
Biomed Res. 2011; 32: 407-11
Display abstract
Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is one of the most important enzymes in the renin-angiotensin system, a major blood pressure control system in mammals. We synthesized novel internally quenched fluorogenic (IQF) substrates for ACE based on the cleavage site of an angiotensin I, introducing N-methyl anthranic acid (Nma) and N(epsilon)-2,4-dinitrophenyl-lysine (Lys(Dnp))at the N- and C-terminal regions. Kinetic parameters of the synthesized IQF substrates Nma-Phe-His-Lys(Dnp) and Nma-His-Pro-Phe-Lys(Dnp)-Pro were compared with those of a common peptide substrate for ACE, hippuryl (Hip)-His-Leu. The k(cat)/K(m) values of Nma-Phe-His-Lys(Dnp), Nma-His-Pro-Phe-Lys(Dnp)-Pro, and Hip-His-Leu were 5.12, 1.90, and 0.80 microM(-1) s(-1) for rabbit lung ACE, and 16.0, 7.36, and 0.30 microM(-1) s(-1) for recombinant human (rh)-ACE, respectively. These results indicate that Nma-Phe-His-Lys(Dnp) is an excellent substrate for rh-ACE. Carboxypeptidase Y also hydrolyzed Nma-Phe-His-Lys(Dnp) efficiently with K(m), k(cat), and k(cat)/K(m) values of 60.2 microM, 105 s(-1), and 1.74 microM(-1) s(-1), respectively. On the other hand, carboxypeptidase B did not hydrolyze IQF substrates. The newly developed IQF substrate, Nma-Phe-His-Lys(Dnp), is a valuable tool for ACE and carboxypeptidase studies.

Bogdanovic X, Singh RK, Hentschke J, Gudmundsdottir BK, Hinrichs W
Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenes.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009; 65: 695-7
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Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P2(1)2(1)2(1)) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 A, beta = 109.3 degrees for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 A, beta = 112.4 degrees for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 A. The crystals of the two different mutants diffracted X-rays beyond 2.0 A resolution.

Caliandro R, Carrozzini B, Cascarano GL, Giacovazzo C, Mazzone AM, Siliqi D
EDM-DEDM and protein crystal structure solution.
Acta Crystallogr D Biol Crystallogr. 2009; 65: 477-84
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Electron-density modification (EDM) procedures are the classical tool for driving model phases closer to those of the target structure. They are often combined with automated model-building programs to provide a correct protein model. The task is not always performed, mostly because of the large initial phase error. A recently proposed procedure combined EDM with DEDM (difference electron-density modification); the method was applied to the refinement of phases obtained by molecular replacement, ab initio or SAD phasing [Caliandro, Carrozzini, Cascarano, Giacovazzo, Mazzone & Siliqi (2009), Acta Cryst. D65, 249-256] and was more effective in improving phases than EDM alone. In this paper, a novel fully automated protocol for protein structure refinement based on the iterative application of automated model-building programs combined with the additional power derived from the EDM-DEDM algorithm is presented. The cyclic procedure was successfully tested on challenging cases for which all other approaches had failed.

Cohen SX et al.
ARP/wARP and molecular replacement: the next generation.
Acta Crystallogr D Biol Crystallogr. 2008; 64: 49-60
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Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information.

Shen MH et al.
Purification, characterization, and cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia.
J Microbiol Biotechnol. 2007; 17: 1271-83
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A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and 35 degrees C, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the A alpha-chain and the B beta-chain over the gamma-chain. Enzyme activity was enhanced by the addition of Ca2+, Zn2+, and Mg2+ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.

Yang BK, Gu YA, Jeong YT, Song CH
Anti-complementary Activities of Exo- and Endo-biopolymer Produced by Submerged Mycelial Culture of Eight Different Mushrooms.
Mycobiology. 2007; 35: 145-9
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The Elfvingia applanata (EA), Hericium erinaceum (HE),Grifola frondosa (GF), Pholiota nameko (PN), Pleurotus eryngii (PE), Trametes suaveolens (TS), Fomes fomentarius (FF), and Inonotus obliquus (IO) could produce the endo- (EN) and exo-biopolymer (EX) in submerged culture. The highest anti-complementary activity of the EN was exhibited by PN (49.1%), followed by HE (38.6%), TS (37.0%),and FF (33.0%),whereas the high activity of the EX was found with GF (59.8%),followed by HE (36.3%),TS (30.8%),and IO (28.8%). The EN of P. nameko (EN-PN) and EX of G. frondosa (EX-GF) were found to contain 78.6% and 41.2% carbohydrates, while 21.4% and 58.8% protein, respectively. The sugar and amino acid compositions of EN-PN and EX-GF were also analyzed in detail.

Ruzza P et al.
Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.
J Pept Sci. 2006; 12: 455-61
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Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.

Minor W, Cymborowski M, Otwinowski Z, Chruszcz M
HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes.
Acta Crystallogr D Biol Crystallogr. 2006; 62: 859-66
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A new approach that integrates data collection, data reduction, phasing and model building significantly accelerates the process of structure determination and on average minimizes the number of data sets and synchrotron time required for structure solution. Initial testing of the HKL-3000 system (the beta version was named HKL-2000_ph) with more than 140 novel structure determinations has proven its high value for MAD/SAD experiments. The heuristics for choosing the best computational strategy at different data resolution limits of phasing signal and crystal diffraction are being optimized. The typical end result is an interpretable electron-density map with a partially built structure and, in some cases, an almost complete refined model. The current development is oriented towards very fast structure solution in order to provide feedback during the diffraction experiment. Work is also proceeding towards improving the quality of phasing calculation and model building.

Depristo MA, de Bakker PI, Johnson RJ, Blundell TL
Crystallographic refinement by knowledge-based exploration of complex energy landscapes.
Structure. 2005; 13: 1311-9
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Although X-ray crystallography remains the most versatile method to determine the three-dimensional atomic structure of proteins and much progress has been made in model building and refinement techniques, it remains a challenge to elucidate accurately the structure of proteins in medium-resolution crystals. This is largely due to the difficulty of exploring an immense conformational space to identify the set of conformers that collectively best fits the experimental diffraction pattern. We show here that combining knowledge-based conformational sampling in RAPPER with molecular dynamics/simulated annealing (MD/SA) vastly improves the quality and power of refinement compared to MD/SA alone. The utility of this approach is highlighted by the automated determination of a lysozyme mutant from a molecular replacement solution that is in congruence with a model prepared independently by crystallographers. Finally, we discuss the implications of this work on structure determination in particular and conformational sampling and energy minimization in general.

Sekar K et al.
A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A2 at 1.6 A resolution using sulfur-SAS at 1.54 A wavelength.
Acta Crystallogr D Biol Crystallogr. 2004; 60: 1586-90
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The crystal structure of the triple mutant K53,56,120M of bovine pancreatic phospholipase A(2) has been redetermined using sulfur single-wavelength anomalous scattering. The synchrotron data were collected at lambda = 1.54 A and the crystal diffracted to 1.6 A resolution. The program SOLVE was used to locate the heavy atoms and to estimate the initial phases and the resulting map was then subjected to RESOLVE. The output of 455 non-H atoms, including 12 S atoms, one calcium ion and one chloride ion, were then subjected to ARP/wARP followed by REFMAC. With the improved phases, the automatic model building successfully built more than 85% of the 123 residues, excluding the N- and C-terminal residues. The final crystallographic R factor is 17.7% (R(free) = 21.7%). The refined model consists of 954 non-H protein atoms, 165 water O atoms, three 2-methyl-2,4-pentanediol (MPD) molecules, one calcium ion and one chloride ion. The present work is yet another example that shows the utility of single-wavelength anomalous scattering data for solving a protein structure.

Bertini I, Calderone V, Fragai M, Luchinat C, Mangani S, Terni B
Crystal structure of the catalytic domain of human matrix metalloproteinase 10.
J Mol Biol. 2004; 336: 707-16
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The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

Juskowiak GL, Stachel SJ, Tivitmahaisoon P, Van Vranken DL
Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions.
J Am Chem Soc. 2004; 126: 550-6
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Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with intracellular conditions, may serve as fluorescent tags for proteins or as fluorescent biomaterials.

Mooers BH, Matthews BW
Use of an ion-binding site to bypass the 1000-atom limit to structure determination by direct methods.
Acta Crystallogr D Biol Crystallogr. 2004; 60: 1726-37
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Proteins with more than 1000 non-H atoms and without heavy-atom prosthetic groups are very difficult to solve by ab initio direct methods. T4 lysozyme is being used to explore these limits. The protein has 1309 non-H atoms, seven S atoms, no disulfide bonds and no heavy-atom prosthetic group. It is recalcitrant to structure determination by direct methods using X-ray diffraction data to 0.97 A. It is shown here that it is possible to obtain a truly ab initio structure determination of a variant of the protein that has an Rb+ (Z = 37) binding site. Using diffraction data to 1.06 A resolution, the direct-methods programs SIR2002 and ACORN independently solved the structure in about 20 h. The bound Rb+, which contributes about 1.7% of the total scattering, does not appear to distort the structure or to inhibit refinement (R factor 12.1%). The phases obtained via SIR2002 or ACORN are in good agreement with those from a reference structure obtained from conventional molecular-substitution and refinement procedures (average error in the figure-of-merit-weighted phases of less than 25 degrees). Thus, proteins with more than 1000 atoms that include halide-binding or other such sites may be amenable to structure determination by ab initio direct methods. The direct-methods approaches are also compared with structure determination via use of the anomalous scattering of the Rb+ ion. As shown by examples, high-resolution structures determined by direct methods can be useful in highlighting regions of strain in the protein, including short hydrogen bonds and non-planar peptide groups.

Kudo N, Yasumasu S, Iuchi I, Tanokura M
Crystallization and preliminary X-ray analysis of HCE-1, a hatching enzyme of medaka fish, Oryzias latipes.
Acta Crystallogr D Biol Crystallogr. 2004; 60: 725-6
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The hatching enzyme of medaka fish, high choriolytic enzyme (HCE-1; MW = 22.7 kDa), was crystallized by the hanging-drop vapour-diffusion method using PEG 10 000 as the precipitant. The hatching enzyme is a metalloproteinase which is secreted from the embryo at the time of hatching. The crystals diffracted X-rays to beyond 1.34 A resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 99.0, b = 30.4, c = 79.6 A, beta = 123.6 degrees. The crystal contains one molecule in an asymmetric unit (VM = 2.2 A3 Da-1) and has a solvent content of 43.7%.

Lecomte C, Guillot B, Muzet N, Pichon-Pesme V, Jelsch C
Ultra-high-resolution X-ray structure of proteins.
Cell Mol Life Sci. 2004; 61: 774-82
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The constant advances in synchrotron radiation sources and crystallogenesis methods and the impulse of structural genomics projects have brought biocrystallography to a context favorable to subatomic resolution protein and nucleic acid structures. Thus, as soon as such precision can be frequently obtained, the amount of information available in the precise electron density should also be easily and naturally exploited, similarly to the field of small molecule charge density studies. Indeed, the use of a nonspherical model for the atomic electron density in the refinement of subatomic resolution protein structures allows the experimental description of their electrostatic properties. Some methods we have developed and implemented in our multipolar refinement program MoPro for this purpose are presented. Examples of successful applications to several subatomic resolution protein structures, including the 0.66 angstrom resolution human aldose reductase, are described.

Lario PI, Sampson N, Vrielink A
Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.
J Mol Biol. 2003; 326: 1635-50
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The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.

Retailleau P, Prange T
Phasing power at the K absorption edge of organic arsenic.
Acta Crystallogr D Biol Crystallogr. 2003; 59: 887-96
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Single/multiple-wavelength anomalous dispersion (SAD/MAD) experiments were performed on a crystal of an organic arsenic derivative of hen egg-white lysozyme. A para-arsanilate compound used as a crystallizing reagent was incorporated into the ordered solvent region of the lysozyme molecule. Diffraction data were collected to high resolution (

Yan ZH, Ren KJ, Wang Y, Chen S, Brock TA, Rege AA
Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.
Anal Biochem. 2003; 312: 141-7
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Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.

George J, Teear ML, Norey CG, Burns DD
Evaluation of an imaging platform during the development of a FRET protease assay.
J Biomol Screen. 2003; 8: 72-80
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Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from biologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH(2) is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMT-based fluorescence plate reader and at the same time evaluated a charged couple device (CCD)-based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens.

Watanabe L, Rucavado A, Kamiguti A, Theakston RD, Gutierrez JM, Arni RK
Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom.
Acta Crystallogr D Biol Crystallogr. 2002; 58: 1034-5
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BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 A.

Yao JX
ACORN in CCP4 and its applications.
Acta Crystallogr D Biol Crystallogr. 2002; 58: 1941-7
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ACORN is a comprehensive and efficient phasing procedure for the determination of protein structures when atomic resolution data are available. Reliable phases can be developed from a fragment composed of a small percentage (less than 5%) of the scattering matter of the unit cell. For example, ACORN has been used to solve a structure of 1093 atoms from only one S atom. The map from ACORN typically reveals the 90% of whole structure. The initial model can be automatically built using ARP/wARP or QUANTA. ACORN can also be used to determine substructures of heavy atoms or anomalously scattering atoms from SAD or MAD data at much lower resolutions such as 3.5 A. Some test results of using ACORN on known structures are presented here. ACORN has solved four new protein structures by users in the UK and France, and some results kindly provided by them are described in this paper.

Coste F, Shepard W, Zelwer C
Description of ordered solvent molecules in a platinated decanucleotide duplex refined at 1.6A resolution against experimental MAD phases.
Acta Crystallogr D Biol Crystallogr. 2002; 58: 431-40
Display abstract
Accurate experimental phases derived from a MAD experiment may be useful to enable the identification of solvent molecules during the course of an atomic parameter refinement. The structure of a double-stranded DNA decanucleotide bearing a cisplatin interstrand cross-link at 1.6A resolution, whose phases were first determined experimentally using the L(III) edge of the Pt atom, was refined by various methods. The previously published structure resulted from a least-squares refinement using the structure-factor magnitudes and stereochemical restraints (program SHELX). In this paper, these previous results are compared with a model obtained by the likelihood-maximization method (program REFMAC) which allows the combination of the observed magnitudes with experimental MAD phases. This solution corresponded to a lower R(free) (18.8 compared with 20.3%), a lower R factor and accounted for 135 water molecules and one spermine molecule collected by the program wARP during refinement. The previously published SHELX solution exhibited no spermine molecule and accounted for 92 water molecules, only 74 of which are also present in the model obtained with the MAD phases. In order to verify that these improvements were actually related to the use of the MAD phases, the same type of procedure without the MAD phases was applied starting from the initial model. The resulting solution had a higher R(free) (20.3%), which could be related to the loss of 22 water molecules and the addition of 20 new ones. MAD phases therefore seem especially helpful in preventing the model bias which may affect the solvent molecules. All models have in common a hydration cage of nine water molecules which surround the platinum residue. In addition to the spermine molecule, the model obtained with the MAD phases allows description of the water-molecule organization, with reproducible motifs related to the base pairs and to the phosphodiester backbone.

Legler PM, Massiah MA, Mildvan AS
Mutational, kinetic, and NMR studies of the mechanism of E. coli GDP-mannose mannosyl hydrolase, an unusual Nudix enzyme.
Biochemistry. 2002; 41: 10834-48
Display abstract
GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608). Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations. Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10(2.2)-fold, increased the dissociation constant of Mn2+ from the ternary E-Mn2+-GDP complex 3-fold, increased the K(m)Mg2+ 20-fold, and decreased the paramagnetic effect of Mn2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn2+. In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn2+ on its side chain -NH2 group. With wild-type GDPMH, the effect of pH on log(kcat/K(m)GDPmann) at 37 degrees C showed an ascending limb of unit slope, followed by a plateau yielding a pK(a) of 6.4, which increased to 6.7 +/- 0.1 in the pH dependence of log(kcat). The general base catalyst was identified as a neutral His residue by the DeltaH(ionization) = 7.0 +/- 0.7 kcal/mol, by the increase in pK(a) with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10(3.4)-fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role. The H88Q mutant showed a 10(2.3)-fold decrease in kcat, a 4.4-fold increase in K(m)GDPmann, and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole HdeltaC, H(epsilon)C, N(delta), and N(epsilon) resonances of the four histidines and defined their protonation states. The pK(a) of His-124 (6.94 +/- 0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/- 0.20). The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold. The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.

Mukherjee M, Maiti S, Ghosh S, Woolfson MM
A direct-method ab initio phasing of a protein, cupredoxin amicyanin, at 1.31 A resolution.
Acta Crystallogr D Biol Crystallogr. 2001; 57: 1276-80
Display abstract
The direct-methods program MULTAN88 has been applied successfully to redetermine the structure of a protein, cupredoxin amicyanin, containing 808 non-H atom sites, one Cu atom and 132 ordered water molecules in the asymmetric unit using data at 1.31 A resolution. Starting with initially random phases, useful phase sets selected by figures of merit could be obtained from multiple trials. The E maps corresponding to the best eight phase sets in order of combined figures of merit (CFOM2) revealed a distorted tetrahedral geometry around the Cu site. The phase estimates from the metal and a few neighbouring atoms in the initial E map corresponding to the set with the highest CFOM2 could be improved by the density-modification procedure PERP and led to an interpretable electron-density map.

Bond CS, Shaw MP, Alphey MS, Hunter WN
Structure of the macrocycle thiostrepton solved using the anomalous dispersion contribution of sulfur.
Acta Crystallogr D Biol Crystallogr. 2001; 57: 755-8
Display abstract
The structure of a tetragonal crystal form of thiostrepton has been solved using the anomalous dispersive effects of five S atoms from high-redundancy data collected to 1.33 A resolution at the Cu Kalpha wavelength. Data measured to 1.02 A resolution with a synchrotron source were used for refinement. Details of the molecular structure, intramolecular and intermolecular interactions are given.

Harris TK, Wu G, Massiah MA, Mildvan AS
Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase.
Biochemistry. 2000; 39: 1655-74
Display abstract
The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M(2+) complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn(2+) at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn(2+), respectively, than the wild type enzyme as detected by 1/T(1) values of water protons, consistent with the loss of a metal ligand. However, the K(m) values of Mg(2+) and Mn(2+) indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M(2+) complexes. Two-dimensional (1)H-(15)N HSQC and three-dimensional (1)H-(15)N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in (1)H-(15)N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. (1)H-(15)N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. (ABSTRACT TRUNCATED)

Perrakis A, Morris R, Lamzin VS
Automated protein model building combined with iterative structure refinement.
Nat Struct Biol. 1999; 6: 458-63
Display abstract
In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

White IJ, Lawson J, Williams CH, Hooper NM
A continuous fluorometric assay for leukotriene D4 hydrolase.
Anal Biochem. 1999; 268: 245-51
Display abstract
A fluorogenic substrate for assay of leukotriene D4 hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acid d, l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2, 4-dinitrophenyl (DNP) group. The compound is epsilon-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing a d-amino acid in the C-terminal position. epsilon-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, with Km value of 370 microoffUnder the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of epsilon-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase.

Mukherjee M
Ab initio structure determination of a small protein, rubredoxin, by direct methods.
Acta Crystallogr D Biol Crystallogr. 1999; 55: 820-5
Display abstract
The direct-methods program SAYTAN has been applied successfully to a known protein, rubredoxin, which contains 52 amino-acid residues including an FeS4 unit, a sulfate ion and 102 solvent water molecules. Starting with initially random phases, useful sets can be obtained from multiple trials and selected by figures of merit at different resolutions. Phase extension followed by weighted Fourier recycling reveals a recognizable structure of rubredoxin. The model is refined against 1 A resolution data to an R factor of 14.5% using the program SHELXL93.

Choi KH, Mazurkie AS, Morris AJ, Utheza D, Tolan DR, Allen KN
Structure of a fructose-1,6-bis(phosphate) aldolase liganded to its natural substrate in a cleavage-defective mutant at 2.3 A(,).
Biochemistry. 1999; 38: 12655-64
Display abstract
Class I fructose-1,6-bis(phosphate) aldolase is a glycolytic enzyme that catalyzes the cleavage of fructose 1,6-bis(phosphate) through a covalent Schiff base intermediate. Although the atomic structure of this enzyme is known, assigning catalytic roles to the various enzymic active-site residues has been hampered by the lack of a structure for the enzyme-substrate complex. A mutant aldolase, K146A, is unable to cleave the C3-C4 bond of the hexose while retaining the ability to form the covalent intermediate, although at a greatly diminished rate. The structure of rabbit muscle K146A-aldolase A, in complex with its native substrate, fructose 1,6-bis(phosphate), is determined to 2.3 A resolution by molecular replacement. The density at the hexose binding site differs between subunits of the tetramer, in that two sites show greater occupancy relative to the other two. The hexose is bound in its linear, open conformation, but not covalently linked to the Schiff base-forming Lys-229. Therefore, this structure most likely represents the bound complex of hexose just after hemiketal hydrolysis and prior to Schiff base formation. The C1-phosphate binding site involves the three backbone nitrogens of Ser-271, Gly-272, and Gly-302, and the epsilon-amino group of Lys-229. This is the same binding site previously found for the analogous phosphate of the product DHAP. The C6-phosphate binding site involves three basic side chains, Arg-303, Arg-42, and Lys-41. The residues closest to Lys-229 were relatively unchanged in position when compared to the unbound wild-type structure. The major differences between the bound and unbound enzyme structures were observed in the positions of Lys-107, Arg-303, and Arg-42, with the greatest difference in the change in conformation of Arg-303. Site-directed mutagenesis was performed on those residues with different conformations in bound versus unbound enzyme. The kinetic constants of these mutant enzymes with the substrates fructose 1, 6-bis(phosphate) and fructose 1-phosphate are consistent with their ligand interactions as revealed by the structure reported here, including differing effects on k(cat) and K(m) between the two substrates depending on whether the mutations affect C6-phosphate binding. In the unbound state, Arg-303 forms a salt bridge with Glu-34, and in the liganded structure it interacts closely with the substrate C6-phosphate. The position of the sugar in the binding site would require a large movement prior to achieving the proper position for covalent catalysis with the Schiff base-forming Lys-229. The movement most likely involves a change in the location of the more loosely bound C6-phosphate. This result suggests that the substrate has one position in the Michaelis complex and another in the covalent complex. Such movement could trigger conformational changes in the carboxyl-terminal region, which has been implicated in substrate specificity.

Van Asselt EJ, Perrakis A, Kalk KH, Lamzin VS, Dijkstra BW
Accelerated X-ray structure elucidation of a 36 kDa muramidase/transglycosylase using wARP.
Acta Crystallogr D Biol Crystallogr. 1998; 54: 58-73
Display abstract
The X-ray structure of the 36 kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 A resolution. Subsequently, before any model building was carried out, phases were extended to 1.7 A resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases. The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened. The structure of the soluble lytic transglycosylase was refined at 1.7 A resolution with X-PLOR to a final crystallographic R factor of 18.9%. Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher. Furthermore, setting aside 5% of the data for an Rfree test set had a negative effect on the phase improvement. The mean WwARP, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the Rfree factor to judge different phase improvement protocols. The elongated Slt35 structure has three domains named the alpha, beta and core domains. The alpha domain contains mainly alpha-helices, while the beta domain consists of a five-stranded antiparallel beta-sheet flanked by a short alpha-helix. Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E. coli. The putative active site is at the bottom of a large deep groove in the core domain.

Perrakis A, Sixma TK, Wilson KS, Lamzin VS
wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models.
Acta Crystallogr D Biol Crystallogr. 1997; 53: 448-55
Display abstract
wARP is a procedure that substantially improves crystallographic phases (and subsequently electron-density maps) as an additional step after density-modification methods such as solvent flattening and averaging. The initial phase set is used to create a number of dummy atom models which are subjected to least-squares or maximum-likelihood refinement and iterative model updating in an automated refinement procedure (ARP). Averaging of the phase sets calculated from the refined output models and weighting of structure factors by their similarity to an average vector results in a phase set that improves and extends the initial phases substantially. An important requirement is that the native data have a maximum resolution beyond approximately 2.4 A. The wARP procedure shortens the time-consuming step of model building in crystallographic structure determination and helps to prevent the introduction of errors.

Yonezawa H, Uchikoba T, Kaneda M, Izumiya N
Sensitive fluorometric assay for the activity of chymosin.
Int J Pept Protein Res. 1996; 47: 56-61
Display abstract
Fluorogenic substrates for chymosin [Dns-Leu-Ser-Phe-Trp-Ala-Leu-OCH2Py (I), Dns-Leu-Ser-Phe-Met-Trp-Leu-OCH2Py (II), Dns-Leu-Ser-Leu-Trp-Ala-Leu-OCH2Py (III), Dns-Leu-Ala-Phe-Trp-Ala-Leu-OCH2Py (IV), Dns-Leu-Ser-Phe-Leu-Ala-Leu-OCH2Py (V) and Dns-Leu-Ser-Phe-Phe-Ala-Leu-OCH2Py (VI)] were synthesized by a solution method. The obtained substrates I-VI were cleaved specifically (between the Phe and Trp residues for substrates I and IV, the Phe and Met residues for substrate II, the Leu and Trp residues for substrate III, the Phe and Leu residues for substrate V, and the Phe and Phe residues for substrate VI) by chymosin. The fluorescence of substrates I-IV (345 nm) increased with their hydrolysis, and hydrolysis rates were obtained by measuring the increase in fluorescence. The minimum detectable chymosin concentrations for substrates I and IV were about 1 nM; those for substrates II and III were about 4 and 2 nM. This assay method is very sensitive, and it is possible to determine the chymosin activity rapidly and easily. Substrates I and IV-VI were hydrolyzed by chymosin two times faster than substrates II and III. The effect of the amino-acid residues of the substrates on the hydrolysis rate is discussed.

Soleilhac JM, Cornille F, Martin L, Lenoir C, Fournie-Zaluski MC, Roques BP
A sensitive and rapid fluorescence-based assay for determination of tetanus toxin peptidase activity.
Anal Biochem. 1996; 241: 120-7
Display abstract
The light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis. The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain. In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method. The fluorescent substrate [Pya88] S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively). One of the two metabolites formed by the action of TeNT L chain, [Pya88] S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence. This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC). It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed.

Kaliberda EN, Rumsh LD
[Preparation of specific substrates for glycopeptidamidase A and kinetics of their enzymatic hydrolysis].
Bioorg Khim. 1995; 21: 28-32
Display abstract
Glycopeptides were obtained by the pepsin (glycononapeptide) and trypsin (glycohexapeptide) hydrolysis of ovalbumin. A method (HPLC) of the glycopeptidamidase activity determination with a fluorescent-labelled (Dns) glycopeptides is described, and kinetic constants of hydrolysis of specific substrates by glycopeptidamidase A are determined. Dns(O-Dns)Tyr-Asn(CHO)-Leu-Thr-Ser-Val is shown to be a specific substrate of the glycopeptidamidase A.

Jean F, Boudreault A, Basak A, Seidah NG, Lazure C
Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor.
J Biol Chem. 1995; 270: 19225-31
Display abstract
The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.

Dohmae N, Hayashi K, Miki K, Tsumuraya Y, Hashimoto Y
Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies.
Biosci Biotechnol Biochem. 1995; 59: 2074-80
Display abstract
A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn(2+)-depleted ProB can regain the activity with Zn2+, Co2+, or Mn2+. Specific cleavage of Pro29-Lys30 in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the alpha-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn2+ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.

Stein RL, Chen Z, Melandri F
Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin.
Biochemistry. 1995; 34: 12616-23
Display abstract
We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angliker H, Neumann U, Molloy SS, Thomas G
Internally quenched fluorogenic substrate for furin.
Anal Biochem. 1995; 224: 409-12
Display abstract
A new substrate for furin, Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH, has been synthesized and characterized. The peptide is an internally quenched fluorogenic substrate. The kinetic parameters are Km = 3.8 microM, kcat = 29.3 s-1, and kcat/KM = 7,710,000 M-1 s-1. The substrate is efficiently cleaved by furin; its kcat/KM value is over 2000-fold higher than that of the commonly used substrate Boc-Arg-Val-Arg-Arg-AMC.

Nonaka T, Ishikawa H, Tsumuraya Y, Hashimoto Y, Dohmae N
Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa.
J Biochem. 1995; 118: 1014-20
Display abstract
A zinc-metalloendopeptidase, MEP, capable of catalyzing specific cleavage of acyl-lysine bonds (-X-Lys-) in polypeptides has been purified 212-fold in a yield of 24.7% from the fruiting bodies of Grifola frondosa, which is a popular edible mushroom called "MAITA-KE" in Japan. The purified enzyme consists of a single polypeptide chain with an apparent molecular mass of 20 kDa and a pI value of 7.46, contains 1 atom of zinc/molecule and can be inactivated with EDTA or 1,10-phenanthroline. Treatment of MEP with EDTA affords an apoenzyme, whose activity can be fully restored by the addition of Mn2+, Zn2+, Ca2+, or Co2+. Prominent features of MEP are its remarkable heat stability and its high affinity for beta-D-glucans and chitin. It hydrolyzes proteins maximally at pH 9-10, liberating only lysylpeptides. Polylysine and lysine copolymers with alanine, phenylalanine, or glutamic acid can serve as good substrates. Lysylalanine was liberated from bovine insulin and its oxidized B chain by the action of MEP. Mass spectrometric analysis by Frit-FAB MS of the fragments generated from horse heart cytochrome c presented unambiguous evidence to corroborate the specificity of MEP for acyl-lysine bonds.

Welch AR, Holman CM, Browner MF, Gehring MR, Kan CC, Van Wart HE
Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate.
Arch Biochem Biophys. 1995; 324: 59-64
Display abstract
Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.

Pennington MW, Thornberry NA
Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.
Pept Res. 1994; 7: 72-6
Display abstract
Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of the molecule. Upon cleavage of DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (DABCYL-ICE-EDANS), an increase in fluorescence is observed at the EDANS emission wavelength of 490 nm, permitting a continuous assay of ICE that is useful in the screening of inhibitory compounds. The Km and kcat results for hydrolysis of DABCYL-ICE-EDANS by ICE were 11.4 +/- 1.6 microM and 0.79 +/- 0.4 s-1. The second order rate constant for hydrolysis of this substrate (kcat/Km = 7.0 +/- 1.3 x 10(4) M-1 s-1) is comparable to that for the cleavage of the previously described fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-AMC (6.4 x 10(4) M-1 s-1).

Chakrabarti AC, Clark-Lewis I, Cullis PR
Influence of charge, charge distribution, and hydrophobicity on the transport of short model peptides into liposomes in response to transmembrane pH gradients.
Biochemistry. 1994; 33: 8479-85
Display abstract
Previous work [Chakrabarti et al. (1992) Biophys. J. 61, 228-234] has shown that basic amino acids and peptides, in which the C-terminal carboxyl groups have been modified to form amides or methyl esters, can be rapidly and efficiently accumulated into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients (delta pH, inside acidic). In this work, the ability of small (di and tri) peptides, composed exclusively of basic (lysine) and hydrophobic (tryptophan) amino acids, to accumulate into LUV systems in response to delta pH has been investigated. In the case of the dipeptides Trp-Lys-amide and Lys-Trp-amide, remarkable differences in the rate constants associated with net transport were observed. In EPC:cholesterol LUV systems exhibiting a delta pH of 3 units (pHi = 4.0; pHo = 7.0), for example, the rate constant for the uptake of Lys-Trp-amide is some 5 x 10(3) faster than for Trp-Lys-amide. Activation energies associated with the uptake also varied from 24 (Lys-Trp) to 29 kcal/mol (Trp-Lys). Related effects were observed for the tripeptides composed of one lysine and two tryptophan residues; however, the differences in rate constants were less sensitive to amino acid sequence. It is concluded that different charge distributions in short peptides of identical amino acid composition can strongly influence the ability of these groups to associate with and permeate across lipid bilayers. These observations may have relevance to the ability of basic peptides, such as signal sequences and peptide hormones, to translocate across biological membranes.

Gershkovich AA
[Fluorescent substrates of proteolytic enzymes with internal quenching of fluorescence].
Ukr Biokhim Zh (1978). 1994; 66: 10-29
Display abstract
The design and application of a successfully developed type of fluorogenic substrates for proteolytic enzymes are discussed. Proteolytic enzymes play a key role not only in the regulation of cellular protein turnover but also in the control of many other physiological functions. Substrates consisting of the appropriate peptides with chromophore or fluorophore at the C-termini are often employed to detect protease activity. Proteases exhibit the primary specificity to the particular amino acid residue and hydrolyze the following peptide bond. However, it is impossible to investigate the specific recognition of the other side of a scissible bond. Intramolecularly quenched fluorescent substrates are very useful instruments for research and medical purpose due to their high sensitivity and selectivity to proteases.

Nagase H, Fields CG, Fields GB
Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3).
J Biol Chem. 1994; 269: 20952-7
Display abstract
Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.

Harnois-Pontoni M, Monsigny M, Mayer R
Hydrosoluble fluorogenic substrates for plasmin.
Anal Biochem. 1991; 193: 248-55
Display abstract
New hydrosoluble fluorogenic substrates for plasmin gluconoylpeptidyl-3-amido-9-ethylcarbazole were synthesized. The substitution of the N-terminal end of the peptides by a gluconoyl group prevents the substrates from aminopeptidase degradation and highly increases their hydrosolubility. The substitution of the peptide C-terminal end by a 3-amino-9-ethylcarbazole group leads to substrates suitable for direct fluorometric assay of plasmin present in cell supernatants or in cell lysates. On the basis of the kinetic parameters of the substrate hydrolysis by plasmin, it was found that D amino acids in the P2 position decrease systematically the kinetic constants of the substrates. The L configuration of the P2 amino acid appears therefore as essential in optimum substrates for plasmin.

Tisljar U, Knight CG, Barrett AJ
An alternative quenched fluorescence substrate for Pz-peptidase.
Anal Biochem. 1990; 186: 112-5
Display abstract
7-Methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitr oph enyl) is introduced as a new quenched fluorescence substrate for assaying Pz-peptidase (also known as soluble metallo-endopeptidase and endo-oligopeptidase). The value of Km for partially purified Pz-peptidase from rat muscle was 8.6 microM. High protein concentrations did not interfere with the assay, so that for the first time continuous assays of Pz-peptidase in crude tissue extracts became possible.

Baskova IP, Timokhina EA, Nikonov GI, Stepanov VM
[Hydrolysis of the isopeptide epsilon-(gamma-glutamyl)-lysine by destabilase from the medicinal leech Hirudo medicinalis].
Biokhimiia. 1990; 55: 771-5
Display abstract
Using amino acid analysis, the ability of destabilize to hydrolyze the epsilon-(gamma-Glu)-Lys isopeptide bond was demonstrated. Incubation of the epsilon-(gamma-Glu)-Lys isopeptide with the enzyme was accompanied by a decrease of the amount of the isopeptide and an increase of equimolar amounts of lysine and glutamic acid. Complete hydrolysis of the isopeptide was observed after 96 hour incubation with destabilize. It was supposed that the isopeptide is a less specific substrate for destabilize compared to L-gamma-Glu-pNA.

Soler DF, Harris RB
Continuous fluorogenic substrates for atrial dipeptidyl carboxyhydrolase. Importance of Ser in the P1 position.
Int J Pept Protein Res. 1988; 32: 35-40
Display abstract
Several N-acyltetrapeptides of the general structure 2-aminobenzoyl-Gly-X-Phe(4-nitro)-Arg were synthesized and tested as substrates for atrial dipeptidyl carboxyhydrolase, an enzyme associated with atrial granules that converts one active atrial natriuretic peptide, atriopeptin II, to another, atriopeptin I. Hydrolysis of the X-Phe(4-nitro) bond generates the 2-aminobenzoyl fluorophore and the increasing fluorescence can be monitored in a continuous assay. Based on the ratio of Vmax/Km as an indication of substrate specificity, peptides containing X = Ser greater than Ala approximately equal to Lys- greater than Asn much greater than Thr approximately equal to Asp. With the exception of the Asn substrate, the Km determined for all the substrates was about the same. Thus, the effect of the P1 residue substitution shows up almost exclusively in Vmax.

Deyrup C, Dunn BM
A new substrate for porcine pepsin possessing cryptic fluorescence properties.
Anal Biochem. 1983; 129: 502-12
Display abstract
A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.

Wayne SI, Fruton JS
Thermolysin-catalyzed peptide bond synthesis.
Proc Natl Acad Sci U S A. 1983; 80: 3241-4
Display abstract
The rates of the thermolysin-catalyzed synthesis of peptides have been determined by means of HPLC. In the condensation of various N-substituted amino acids and peptides with L-leucinanilide, the enzyme exhibits preference for a hydrophobic L-amino acid as the donor of the carbonyl group of the newly formed bond. The presence of another hydrophobic amino acid residue adjacent to the carbonyl-group donor markedly enhances the rate of synthesis. In general, the effect of structural changes in both the carboxyl and amine components of the condensation reaction is in accord with the available data on the primary and secondary specificities of the thermolysin-catalyzed hydrolysis of oligopeptide substrates. A kinetic study of the condensation of benzyloxycarbonyl-L-phenylalanine with various amine components has given data on the apparent kcat and Km values for the entry of the acidic component into the condensation reaction. The results are consistent with the behavior of rapid-equilibrium random bi-reactant systems leading to ternary enzyme-substrate complexes, with a synergistic effect in the binding of the two reactants at the active site. Because the changes in the apparent kcat for the entry of the same acidic component into reaction with different amine components are greater than those in the apparent Km, it is suggested that this synergism is largely expressed at the level of the transition-state complex.

Araujo-Viel MS, Juliano L, Prado ES
The cleavage of the Met-Lys bond in a bradykinin derivative by glandular kallikreins.
Hoppe Seylers Z Physiol Chem. 1981; 362: 337-45
Display abstract
The synthetic tridecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg was used as a model substrate for horse urinary and porcine pancreatic kallikreins. The Met-Lys bond is hydrolyzed selectively by both enzymes. Oxidation of the methionine residue to sulfoxide made the peptide resistant to both kallikreins. Substitution of either the methionine or lysine residues by norleucine led to peptides in which the Nle-Lys or the Met-Nle bonds, respectively, were susceptible to the urinary kallikrein. The esterolytic and Met-Lys bond-splitting activities of both enzymes were inhibited similarly by phenylmethanesulfonyl fluoride. Both activities of the pancreatic kallikrein were inhibited by the chloromethane derivative Ala-Leu-Lys-CH2Cl. Inhibition by benzamidine of Met-Lys hydrolysis by both kallikreins was observed.

Behmoaras T, Toulme JJ, Helene C
Specific recognition of apurinic sites in DNA by a tryptophan-containing peptide.
Proc Natl Acad Sci U S A. 1981; 78: 926-30
Display abstract
We have used fluorescence spectroscopy to study the binding of lysyltryptophyl-alpha-lysine (Lys-Trp-Lys) to DNA modified by dimethyl sulfate before and after depurination and strand breakage. Quenching of tryptophan fluorescence increased upon association of the peptide with modified DNA as compared with native DNA. We have demonstrated that this quenching is related to a preferential stacking of the indole ring with nucleic acid bases in damaged regions. Stacking increased in the following order: methylated DNA less than DNA with strand breaks at apurinic sites much less than apurinic DNA. For apurinic DNA, the overall association constant of Lys-Trp-Lys was increased by more than two orders of magnitude as compared to native DNA. Enhancement of the affinity of the tripeptide for an apurinic site requires the integrity of the phosphodiester bond. Single-strand cleavage at an apurinic site leads to a marked decrease of the association constant. The peptide Lys-Trp-Lys is therefore able to recognize destabilized regions in the vicinity of a lesion and to discriminate between different configurations of the damaged region. These results are discussed with respect to the role that stacking interactions could play in the specificity of recognition of DNA alterations by enzymes involved in DNA repair mechanisms.

Terada S, Kato T, Izumiya N
Synthesis and hydrolysis by pepsin and trypsin of a cyclic hexapeptide containing lysine and phenylalanine.
Eur J Biochem. 1975; 52: 273-82
Display abstract
1. A cyclic hexapeptide, cyclo(-Gly2-Phe2-Gly-Lys-), and the corresponding open-chain hexapeptides, Gly2-Phe2-Gly-Lys and Phe-Gly-Lys-Gly2-Phe, have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined. 2. The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2-Phe2-Gly-Lys, the value of the Michaelis constant for this reaction being Km equals 0.00022 M. 3. The cyclic peptide was not cleaved by pepsin at all, but Gly2-Phe2-Gly-Lys was hydrolyzed rapidly at a Phe-Phe bond; Km equals 0.0091 M. 4. The cyclic peptide inhibits the hydrolysis of Gly2-Phe2-Gly-Lys by pepsin in a linear non-competitive manner, the value of the inhibition constant being Ki equals 0.004 M.

Blaich R
[Aminopeptidases of basidiomycetes. II. Comparison of substrate specificities: (proline)iminopeptidase and (leucine)aminopeptidase of Hapalopilus nidulans--aminopeptidase 53 of Pleurotus ostreatus].
Arch Mikrobiol. 1973; 88: 111-8

Lasota W, Mlodecki H, Wlodarczyk Z
[Content of available lysine in some mushrooms].
Rocz Panstw Zakl Hig. 1968; 19: 459-62

HIRST EL, JONES JK, ROUDIER AJ
Structure of acorn starch.
J Chem Soc. 1948; 70: 1779-83