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)

The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates.
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PMID:Esterase activity of zinc neutral proteases. 0 76

Thei nhibition of the thermolysin catalyzed hydrolysis of FA-Gly-Leu-NH2 and FA-Gly-Phe-NH2 has been reported. The results suggest a model for substrate and inhibitor binding involving the hydrophobic specificity pocket, Arg-203 and Glu-143.
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PMID:Studies on the inhibition of thermolysin. 0 70

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.
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PMID:Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites. 6 52

Purified Japanese monkey pepsinogens I and II contain carbohydrate as a part of the enzyme molecule. By gel filtration on Sephadex G-100, chromatography on DE-32 cellulose, and polyacrylamide disc gel electrophoresis, the carbohydrate moiety could not be separated from the enzyme protein, and the content did not decrease on repeated chromatography. Glycopeptides were obtained by successive digestion of pepsinogens with thermolysin and aminopeptidases and isolated by chromatography on Sephadex G-25 and G-50. Identification and determination of carbohydrate components was performed by paper and gas-liquid chromatographies. The presence of 4 glucosamines, 6 galactoses, 6--8 mannoses, and 8--11 fucoses per molecule of the glycopeptide of both pepsinogens was observed, of which the high content of fucose is especially unique. The molecular weight of the carbohydrate chains should be around 4,000--5,000. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.
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PMID:Monkey pepsinogens and pepsins. III. Carbohydrate moiety of Japanese monkey pepsinogens and the amino acid sequence around the site of its attachment to protein. 10 35

A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.
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PMID:Molecular basis of altered enzyme specificities in a family of mutant amidases from Pseudomonas aeruginosa. 11 34

Synthesis of a series of active N-hydroxysuccinimide esters of aliphatic and aromatic amino acids has yielded a new class of reagents for the covalent modification of proteolytic enzymes such as thermolysin. The activities of aliphatic acyl amino acid thermolysins are from 1.7 to 3.6 times greater than that of the native enzyme when hydrolyzing durylacryloyl-Gly-Leu-NH2, the substrate employed most widely. By comparison, the aromatic acylamino acid derivatives are "superactive," their activities being as much as 70-fold greater. Apparently, the aromatic character of the amino acid introduced is a critical variable in the determination of the functional response. The increased activity is completely restored to that of the native enzyme by deacylation with nucleophiles, such as hydroxylamine, and the rate of restoration of native activity is a function of the particular acyl group incorporated. Preliminary evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified. The functional consequences of successive modification with different reagents, moreover, indicate that each of them reacts with the same protein residue. The competitive inhibitors beta-phenyl-propionyl-Phe and Zn-2+ do not prevent modification with these active esters. Hence, the site(s) of their inhibitory action differ(s) from that at which modification occurs. The structure of the substrate is also a significant variable which determines the rate at which each acyl amino acid thermolysin hydrolyzes peptides. Depending on the particular substrate, the activity of aromatic derivatives can be as much as 400-fold greater than that of the native enzyme, and the resultant activity patterns can be ordered in a series characteristic for each enzyme derivative.
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PMID:Superactivation of thermolysin by acylation with amino acid N-hydroxysuccinimide esters. 23 33

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (similar to 35,000 daltons) is converted to a membrane-bound fragment O' (similar to 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O' are replaced by the membrane-bound fragments F1 (similar to 25,000 daltons), and F2 (similar 9,500 daltons). When 32P-ROS are digested, F2 carries the 32P. Both O' and F1 contain the amino-terminal glycopeptide.
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PMID:The amino- and carboxyl-terminal sequence of bovine rhodopsin. 59 23

A series N-hydroxysuccinimide esters of acylamino acids previously shown to acylate and thereby increase the activity of thermolysin by several orders of magnitude (Blumberg, S., and Vallee, B. L. (1975), Biochemistry 14, 2410) has been used to modify the related neutral proteases from Bacillus subtilis, Bacillus megaterium, and Aeromonas proteolytica. Each of these enzymes is activated to a level characteristic of the particular protein and the particular acyl group incorpporated when monitored with the substrate furylacryloyl-Gly-Leu-NH2. Thus, for the modification of B. megaterium, B. subtilis, and A. proteolytica proteases with Ac-Trp-ONSu, kcat/Km increases 11-, 2.5-, and 18-fold whereas those of the Ac-Phe(4-DnpNH)-ONSu modified enzymes before and after deacylation with hydroxylamine indicate that from 1 to 2 residues are modified. The rate of removal of the Ac-Phe(4-DnpNH) label by 0.1 M hydroxylamine correlates directly with that of the return of native enzymatic activity, at a rate comparable with the rate of deacylation of O-acyltyrosine models. The competitive inhibitors Zn2+ and beta-phenyl-propionyl-Phe do not prevent activation indicating that modification occurs at a site(s) distinct from that at which inhibitors bind. The degree of activation depends also on the substrate employed, generally being greater for substrates which the native enzymes hydrolyze slowly. These data are interpreted to indicate the modification of a residue near the active site, but which serves as a subsite for substrate interaction.
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PMID:Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters. 82 65

The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.
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PMID:Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative. 95 73

The structure of two functional sites in baker's yeast (Saccharomyces cerevisiae) glycogen phosphorylase (EC 2.4 1.1) was determined as part of a study on the evolution of regulatory enzymes. S-Carboxymethylated, MaBH4-reduced 32-P-labeled yeast phosphorylase a was cleaved with CNBr, thermolysin, and pepsin. Peptides labeled with 32-P or carrying the fluorescent pyridoxyl marker were isolated and purified using ion-exchange chromatography and gel filtration. CNBr cleavage yielded a single radioactive phosphopeptide (42 residues long) and one small fluorescent peptide with the unique sequence epsilon-Pxy-Lys-Phe-Val-Met. Thermolysin digestion gave rise to one radioactive octapeptide and two fluorescent peptides, 15 and 2 residues long, respectively. From a combination of substractive Edman degradations and digestion with yeast protease C, the sequence of the 32-P-labeled octapeptide was established. Phosphothreonine was identified as the sole phosphorylated amino acid, giving the following structure for the site involved in the covalent regulation of yeast phosphorylase: Leu-Thr(P) -Gly-Phe-Leu-Pro-Gln-Glu. The two fluorescent thermolytic peptides, together with two additional pyridoxyl peptides isolated after peptic digestion of the enzyme yielded the following sequence around the site binding pyridoxal-5'-P, the cofactor essential for phosphorylase activity: Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys(P Pxy)-Phe-Val-Met. While the phosphorylated site bears no resemblance to the site of covalent control in vertebrate phosphorylases, the pyridoxal-P binding site in the yeast enayme displays remarkable homologies with its animal counterparts; the finding that 14 out of 18 amino acids are identical strongly suggests that the cofactor must be directly involved in catalysis.
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PMID:Amino acid sequence of two functional sites in yeast glycogen phosphorylase. 109 46


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