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)

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

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

1. When thermolysin was treated with a 100-fold molar excess of 2,4,6-trinitrobenzene-1-sulfonate at pH 8.0 and 37 degrees for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity. 2. When treated with a 100- to 1,000-fold molar excess of N-acetylimidazole at pH 6.5 and 23 degrees for 2 h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site. 3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide at pH 6.0 and room temperature for 1 h both in the presence and absence of 8 M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inactivated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8 M urea retarded the inactivation.
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PMID:Studies on thermolysin. I. Effects of chemical modifications on the activity of thermolysin. 84 38

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.
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PMID:Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP. 128 23

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.
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PMID:Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. 249 16

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.
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PMID:Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B. 351 5

Diethylpyrocarbonate treatment of the neutral endopeptidase (EC 3.4.24.11) inhibits both catalytic activity and binding of the inhibitor [3H]-N(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine. The loss of activity can be reversed by hydroxylamine and almost completely prevented by the competitive inhibitor phenylalanyl-leucine suggesting the presence, as in thermolysin, of a histidine residue at the active site. Butanedione treatment also reduces both catalytic activity and [3H] inhibitor binding. Phenylalanyl-leucine completely protects from the butanedione induced loss of activity, providing further evidence for an essential arginine at the active site. In contrast, the tyrosine modifying agent N-acetylimidazole has no apparent effect on enzyme activity.
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PMID:Presence of a histidine at the active site of the neutral endopeptidase-24.11. 353 67

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin. 643 41

The peptide alpha 1(III)-CB9 was prepared and purified from human liver, and its amino acid sequence was determined. Automated Edman degradation of the intact peptide and peptides derived from selective cleavage with hydroxylamine and digestions with trypsin, thermolysin, and Staph V8 protease enabled determination of the complete amino acid sequence. The peptide alpha 1(III)-CB9 represents the COOH terminus of the helical (pepsin-resistant) portion of type III collagen and terminates in a Cys-Cys sequence responsible for the intramolecular disulfide cross-linkages with other chains. The present work completes the entire amino acid sequence of the helical (pepsin-resistant) portion of human cirrhotic liver type III collagen consisting of peptides alpha 1-(III)-CB3-7-6-1-8-10-2-4-5-9. The COOH terminus of human liver alpha 1(III) contained two additional triplets which, together with the extra triplet at the NH2 terminus in alpha 1(III)-CB3, make the helical portion of type III collagen longer than alpha 1(I) by nine residues (three Gly-X-Y triplets). The helical region of human liver type III collagen, therefore, consists of 1023 amino acids or 341 triplets.
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PMID:Covalent structure of collagen: amino acid sequence of alpha 1(III)-CB9 from type III collagen of human liver. 701 80

Two tandem cysteine residues in the carboxyl-terminal region of rhodopsin have been shown to be covalently linked to palmitate via thioester bonds (Ovchinnikov, Y. A., et al. (1988) FEBS Lett. 230, 1-5). We have synthesized a fluorescent analogue of palmitoyl coenzyme A (16-(9-anthroyloxy)hexadecanoyl coenzyme A ester) and incorporated the fluorescent derivative of palmitate into the protein in high yield (> 40%) through pretreatment of bovine rod outer segments with 1 M hydroxylamine and subsequent incubation with the fluorescent label. Covalent incorporation of label into protein was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic digestion of labeled rhodopsin in the disc membrane with papain and thermolysin verified the C-terminal location of the label. Treatment of SDS-solubilized, labeled rod outer segments with 10% beta-mercaptoethanol provided evidence that partial depalmitoylation may induce the formation of rhodopsin aggregates. Labeled, unbleached rhodopsin was purified by chromatography over hydroxyapatite and concanavalin A-agarose and reconstituted into dimyristoylphosphatidylcholine vesicles. SDS gels of the rhodopsin vesicle preparation verified that all unbound fluorescent label had been removed and that the thioester bond linking probe to protein was not labile.
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PMID:Fluorescence labeling of the palmitoylation sites of rhodopsin. 818 Feb 6


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