Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino-acid sequence of tyrosinase from Neurospora crassa (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is reported. This copper-containing oxidase consists of a single polypeptide chain of 407 amino acids. The primary structure was determined by automated and manual sequence analysis on fragments produced by cleavage with cyanogen bromide and on peptides obtained by digestion with trypsin, pepsin, thermolysin, or chymotrypsin. The amino terminus of the protein is acetylated and the single cysteinyl residue 96 is covalently linked via a thioether bridge to histidyl residue 94. The formation and the possible role of this unusual structure in Neurospora tyrosinase is discussed. Dye-sensitized photooxidation of apotyrosinase and active-site-directed inactivation of the native enzyme indicate the possible involvement of histidyl residues 188, 192, 289, and 305 or 306 as ligands to the active-site copper as well as in the catalytic mechanism of this monooxygenase.
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PMID:Amino acid sequence of tyrosinase from Neurospora crassa. 15 Dec 79

Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.
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PMID:Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties. 99 99

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues. 160 44

The three-dimensional structures of (S)-thiorphan and (R)-retro-thiorphan bound to thermolysin have been determined crystallographically and refined to residuals of 0.183 and 0.187 at 1.7-A resolution. Thiorphan [N-[(S)-2-(mercaptomethyl)-1-oxo-3-phenylpropyl]glycine] [HSCH2CH(CH2C6H5)CONHC-H2COOH] and retro-thiorphan [[[(R)-1-(mercaptomethyl)-2-phenylethyl] amino]-3-oxopropanoic acid] [HSCH2CH(CH2C6H5)NHCOCH2COOH] are isomeric thiol-containing inhibitors of endopeptidase EC 24-11 (also called "enkephalinase"). The mode of binding of thiorphan to thermolysin is similar to that of (2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide [Monzingo, A.F., & Matthews, B.W. (1982) Biochemistry 21, 3390-3394] with the inhibitor sulfur atom coordinated to the active site zinc and the peptide portion forming substrate-like interactions with the enzyme. The isomeric inhibitor retro-thiorphan, which differs from thiorphan by the inversion of an amide bond, utilizes very similar interactions with enzyme. Despite the inversion of the -CO-NH- linkage the carbonyl oxygen and amide nitrogen display very similar hydrogen bonding, as anticipated by B.P. Roques et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3178-3182]. These results explain why thermolysin and possibly other zinc endopeptidases such as endopeptidase EC 24-11 fail to discriminate between these retro-inverso inhibitors.
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PMID:Thiorphan and retro-thiorphan display equivalent interactions when bound to crystalline thermolysin. 271 12

The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].
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PMID:Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin. 370 36

Oxygen does not quench the luminescence of either free Tb or of Tb bound to dipicolinate. However, sensitized Tb luminescence in complexes of that ion with elastase, thermolysin, and alpha-amylase is quenched by oxygen at rates that far exceed that with which the intrinsic fluorescence of the proteins is quenched. We infer that this more rapid quenching of Tb luminescence indicates a major role for energy transfer from tryptophan moieties in a triplet excited state.
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PMID:Oxygen quenching of sensitized terbium luminescence in complexes of terbium with small organic ligands and proteins. 618 34

Transpeptidation reactions catalyzed by chymotrypsin, pepsin, leucine aminopeptidase and thermolysin have been studied in heavy oxygen water (H2 18O). The 18O incorporation into the peptide bond of transpeptidation products and into the non-hydrolyzed substrate has been measured. The rates of 18O exchange in the carboxylic groups of N-acetylphenylalanine and leucine, catalyzed by pepsin and leucine aminopeptidase, respectively, have also been determined. These rates have been compared with that of the exchange in the presence of amino compounds which reversibly form amide bonds with the above carboxyl-containing substances. The data obtained show that, in contrast to chymotrypsin, other enzymes studied do not form 'acyl-enzymes' but function by the mechanism of general-base catalysis. In other words, their catalytically active groups promote the abstraction of a proton from the water molecule, which attacks the susceptible bond of the substrate. The structure of intermediate compounds in this type of catalysis and the mechanism of the transpeptidation reaction are discussed.
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PMID:Studies on the mechanisms of action of proteolytic enzymes using heavy oxygen exchange. 679 Feb 82

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.
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PMID:Crystal structures of recombinant 19-kDa human fibroblast collagenase complexed to itself. 803 54

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.
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PMID:Molecular studies of CtpA, the carboxyl-terminal processing protease for the D1 protein of the photosystem II reaction center in higher plants. 870 85

In acute intraocular inflammation, neutrophils release a variety of agents, that are potentially toxic to the surrounding tissues. The reactive oxygen metabolites, including superoxide are among these injurious agents. In the present study, retinal pigment epithelial (RPE) cells were found to inhibit by 70% to 80% the production of superoxide by neutrophils that were stimulated either by the receptor-coupled activator, N-formyl-methionyl-leucyl-phenylalanine, or by the non-receptor-coupled activator, phorbol myristate acetate. The inhibition is effective with a relatively small number of RPE cells (0.65 x 10(5)) mixed in a large pool of neutrophils (7.6 x 10(5)). The transduction of this effect does not require a neutrophil-RPE cell surface contact since the RPE culture supernatants also effectively inhibit the superoxide production. This protein is secreted specifically by cultured and noncultivated intact RPE cells, but not by fibroblasts, corneal epithelial cells or intact choroidal tissues. The protein is not cytotoxic to the neutrophils, and the inhibitory effect occurs in a dose-dependent manner up to the concentration tested. This factor is unrelated to either transforming growth factor-beta, or transferrin. There was no evidence of RPE scavenging of superoxide generated enzymatically by hypoxanthine-xanthine oxidase system, indicating that this factor does not have superoxide dismutase activity. When RPE cells were preincubated with 10 micrograms ml-1 cycloheximide, 60% of the activity was lost, suggesting that a de novo protein synthesis is required for the activity and that the protein is a significant steady-state product of RPE cells. Incubation of the released RPP with thermolysin (10 micrograms ml-1 15 min, 37 degrees C) also eliminated the activity. The degradation of activity by protease and inhibition of RPP activity by cycloheximide, therefore, confirmed the protein nature of the suppressive factor. This protein from RPE cells appears to act directly on the neutrophils, reducing the release of oxygen metabolites during activation, and thus limiting tissue injury during inflammation.
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PMID:A novel retinal pigment epithelial protein suppresses neutrophil superoxide generation. I. Characterization of the suppressive factor. 906 78


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