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

Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M.
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PMID:Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal. 403 Jul 34

Bovine photoreceptor membranes have been treated with proteases to determine the accessibility of rhodopsin to these large, water soluble molecules. The polypeptides that remain associated with the membranous structure after proteolysis were detected by sodium dodecyl sulfate gel electrophoresis. Thermolysin and chymotrypsin degraded rhodopsin (apparent mol wt 35,000-36,000) to fragments of 29,000 and 23,000 apparent mol wt, respectively, without affecting the chromophoric absorption of the molecule or removing the region of the polypeptide carrying carbohydrate. The two fragments were isolated and their amino acid compositions were determined. They do not appear to be more hydrophobic than rhodopsin. Subtilisin, at low concentration and temperature, produced a fragment with the same molecular weight as that produced by thermolysin. At higher concentrations, subtilisin yields major fragments of mol wt 23,000 and 20,000 without affecting the chromophoric absorption. Two intermediate fragments of apparent mol wt 29,000 and 26,000 were detected during the course of this degradation. Carbohydrate is retained by all but the smallest fragment. Bleaching of the photoreceptor pigment did not appreciably alter any of the fragmentation patterns. Trypsin did not alter the molecular weight of rhodopsin under the conditions of this study. Approximately 35-45% of rhodopsin appears to be accessible to the aqueous environment and can be removed without affecting the chromophoric properties of the retinaldehyde-carrying region which remains bound to the membrane.
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PMID:The accessibility of bovine rhodopsin in photoreceptor membranes. 441 32

Gas vesicles, isolated from lysed Halobacterium halobium cells, gave an amino acid analysis which accounted for 78% of the weight, and the balance was mainly salt and water. One percent of tightly bound d-galactose was found, as well as 2% of phosphate that was not released by treatment which promotes beta-elimination, by hydrolytic release of the galactose, by carboxymethylation of lysine, or by alkaline phosphatase digestion. Only a trace of lipid was detected, and it appeared to have a polyisoprenoid structure. The vesicles were not solubilized by extremes of pH, by agents such as urea, guanidine hydrochloride, formic acid, and detergents, or by organic solvents. Succinylation and carboxymethylation gave partial dispersion, but the products were heterogeneous and of high molecular weight. The amino acid composition of vesicles was independent of fragment size. No band was obtained by polyacrylamide gel electrophoresis, with neutral, acidic, and alkaline systems, with or without sodium dodecyl sulfate and urea, before or after chemical modification. No amino terminus was detected. Electrofocusing of a vesicle dispersion showed a major component with a pI of 4.0 and an amino acid composition of the whole vesicles, and a minor band with pI 3.4 which had an amino acid composition different from whole vesicles. Vesicle protein was resistant to digestion by Pronase, trypsin, thermolysin, and papain. The precipitin reaction with rabbit antivesicle serum was not inhibited by galactose or inorganic phosphate. Succinylated and carboxymethylated vesicles cross-reacted with antivesicle serum. Cell lysates contained material which reacted with antiserum, but it was heterogeneous and mainly larger than 5 x 10(6) daltons. Material from nonvacuolated mutants reacted weakly with antiserum, but the amino acid composition of the precipitated antigen was different from that of vesicles and of soluble cross-reacting material from vacuolated cells.
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PMID:Analysis of Halobacterium halobium gas vesicles. 473 10

Proteolytic enzyme activity releasing sialo glycopeptides from 3H-labeled human erythrocyte ghosts was detected in cytotoxic (leukotoxic) culture supernatants from 9 of 12 Pasteurella haemolytica serotypes. Microcrystalline cellulose thin-layer chromatograms of radioactive water-soluble products showed the following two radioactive peaks: a high-mobility minor peak (Rf, 0.54 to 0.74), identified as sialic acid, and a low-mobility major peak (Rf, 0.18 to 0.21), partially characterized as a trichloroacetic acid-soluble, sialic acid-rich fragment with a molecular weight of greater than 3,500, not extractable by chloroform. The sialic acid content of this fragment after treatment with Clostridium perfringens neuraminidase was estimated to be 7.2 X 10(-2) mumol mg-1. The presence of neuraminidase as a separate activity in some culture supernatants was confirmed. It is considered to be responsible for the observed release of free sialic acid. Preliminary studies with the crude enzyme showed that it has a broad pH optimum around pH 7.0 and that activity is not affected by inhibitors of trypsin, chymotrypsin, thermolysin, thio and serine enzymes, nor by an inhibitor of neuraminidase, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. Activity was, however, inhibited by o-phenanthroline at a high concentration after prolonged treatment. The enzyme hydrolyzed glycophorin at a rate four times higher than the rate for casein. Free glycophorin inhibited the enzyme-induced release of radioactive products from 3H-labeled ghosts. It is speculated that the novel enzyme is a neutral protease, probably metal-dependent, with specificity for sialoglycopeptides. The possible relationship of this protease to the previously reported host species-specific leukotoxicity of P. haemolytica and its potential role in virulence is discussed.
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PMID:Proteolysis of sialoglycoprotein by Pasteurella haemolytica cytotoxic culture supernatant. 635 4

The mode of binding of the specific thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan (KI approximately 5 X 10(-8) M) [Maycock, A. L., DeSousa, D. M., Payne, L. G., ten Broeke, J., Wu, M. T., & Patchett, A. A. (1981) Biochem. Biophys. Res. Commun. 102, 963-969] has been determined by X-ray crystallography and refined to an R value of 17.1% at 1.9-A resolution. The inhibitor binds to thermolysin with both oxygens of the N-carboxymethyl group liganded to the zinc to give overall pentacoordination of the metal. The bidentate ligation of the inhibitor differs from the monodentate binding seen previously for carboxylate-zinc interactions in thermolysin and is closer to the bidentate geometry observed for the binding of hydroxamates [Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912-6920]. The geometry of the inhibitor and its interactions with the protein have a number of elements in common with the presumed transition state formed during peptide hydrolysis. The observed zinc ligation supports the previous suggestion that a pentacoordinate intermediate participates in the mechanism of catalysis. However, the alpha-amino nitrogen of the inhibitor is close to Glu-143, suggesting that this residue might accept a proton from an attacking water molecule (as proposed before) and subsequently donate this proton to the leaving nitrogen. By analogy with thermolysin, it is proposed that a related mechanism should be considered for peptide cleavage by carboxypeptidase A.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of N-carboxymethyl dipeptide inhibitors to thermolysin determined by X-ray crystallography: a novel class of transition-state analogues for zinc peptidases. 639 81

Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.
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PMID:An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides. 652 36

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

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.
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PMID:The complete amino acid sequence of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of streptomyces albus G. 682 89

The structure of the complex of thermolysin and the inhibitor (2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide has been determined by X-ray crystallography at a resolution of 1.9 A and refined to a crystallographic residual of 18.4%. The binding of this potent, specific inhibitor to thermolysin (Ki = 7.5 X 10(-7) M) serves as a model for the inhibition of zinc peptidases by substrate-analogue mercaptans. The study shows that the mercaptan inhibitor binds to thermolysin with the sulfur, presumably in the anionic form, tetrahedrally coordinated to the zinc and displacing a water molecule bound to the native enzyme. This is the first direct determination of the mode of binding of a mercaptan inhibitor to a zinc peptidase and confirms the geometry of binding expected on general grounds [Ondetti, M. A., Condon, M. E., Reid, J., Sabo, E. F., Cheung, H. S., & Cushman, D. W. (1979) Biochemistry 18, 1427-1430; Nishino, N., & Powers, J. C. (1979) Biochemistry 18, 4340-4347] and inferred from previous spectroscopic studies [Holmquist, B., & Vallee, B. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6216-6220].
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PMID:Structure of a mercaptan-thermolysin complex illustrates mode of inhibition of zinc proteases by substrate-analogue mercaptans. 705 22

The pH dependences of kcat/Km, kcat and Km of thermolysin-catalyzed hydrolysis of N-furylacryloylglycyl-L-leucinamide (Fua-Gly-LeuNH2) and N-furylacryloylglycyl-L-phenylalaninamide (Fua-Gly-PheNH2) were investigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell-shaped curve with pKa1 = 5.0 and pKa2 = 8.25, at 25 degrees C. Both kcat and Km increased with pH at lower pH and took larger values for Fua-Gly-LeuNH2 than for Fua-Gly-PheNH2 at 25 degrees C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy-L-phenylalanine and L-phenylalanyl-L-leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH. Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non-linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated delta H values of deprotonation for pKa1 and pKa2 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKa1 is too large to be assigned to the carboxylic group of Glu-143, in contrast to the generally accepted view. A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc-coordinated water.
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PMID:pH and temperature dependences of thermolysin catalysis. Catalytic role of zinc-coordinated water. 708 22


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