Thioltrypsin

Yokosawa, Hideyoshi; Ojima, Setsuko; Ishii, Shin‐ichi

doi:10.1093/oxfordjournals.jbchem.a131763

Cited by 45 publications

(18 citation statements)

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“…Finally, replacement of the essential hydroxyl group of serine-119 with the chemically similar thiol group resulted in retention of appreciable levels of activity. A similar pattern has been observed for the serine hydrolytic enzymes 83-lactamase, alkaline phosphatase, trypsin, and subtilisin (26)(27)(28)(29). In what follows, we shall assume that both serine-119 and lysine-156 are directly involved in the chemistry of bond rearrangement.…”

Section: Discussionmentioning

confidence: 53%

Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

Slilaty¹,

Little²

1987

Proc. Natl. Acad. Sci. U.S.A.

214

154

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LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.Escherichia coli LexA and X cI repressors are inactivated in vivo by a specific cleavage reaction that cuts a conserved Ala-Gly bond near the center of the polypeptide chain (1, 2). Cleavage occurs following treatments that damage DNA or inhibit replication (1-3). Inactivation of LexA is rapid and results in derepression of a set of genes, the SOS regulon, largely responsible for DNA repair. Cleavage of the X repressor is much slower than that of LexA, and more severe treatments are required to induce the prophage. Repressor cleavage in vivo requires RecA function; RecA protein is quiescent during normal cell growth but is activated by the inducing treatment to a form that participates in cleavage.Early in vitro studies showed that the role of RecA protein in cleavage is relatively direct (4). At physiological pH, cleavage is dependent on the presence of RecA protein and two cofactors-a nucleoside triphosphate and single-stranded DNA-that activate RecA by forming a ternary complex. For a time, it was tacitly assumed that activated RecA was a conventional protease with a highly specific active site. This view was challenged by the finding (5) that, under different conditions, cleavage of LexA and the X repressor proceeds spontaneously at the same Ala-Gly bond in an intramolecular reaction, termed "autodigestion," that is stimulated by alkaline pH and does not require RecA. Both RecA-dependent cleavage and autodigestion of LexA are inhibited in a mutant protein, LexA3, that also cannot be cleaved in vivo.It was, therefore, proposed that RecA protein is not itself a protease, but rather that it acts indirectly by stimulating the autodigestion reaction. According to this hypothesis, activated RecA is a positive ef...

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Section: Discussionmentioning

confidence: 53%

Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

Slilaty¹,

Little²

1987

Proc. Natl. Acad. Sci. U.S.A.

214

154

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“…A similar loss in activity towards specific substrates has been observed on this transformation of serine proteinases [11][12][13]. The precise reasons, at the molecular level, for this loss are not agreed upon [11,12,[14][15][16][17].…”

Section: Introductionsupporting

confidence: 53%

Inactivation of the thiol RTEM-1 β-lactamase by 6-β-bromopenicillanic acid. Identity of the primary active-site nucleophile

Knap

Pratt

1987

Biochemical Journal

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The thiol RTEM-1 beta-lactamase [Sigal, Harwood & Arentzen (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme. Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate. Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group. These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover. The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm. Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently. Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase.

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“…Experimental simulations of the papain triad in a trypsinlike Ser protease have used direct chemical modification (45) or mutagenic (46) This study proposes a compelling model for the tertiary fold and active-site geometry of the viral Cys proteases that clearly differentiates them from the papain-like class of cellular Cys proteases. Previous analyses (16-19, 22, 24) have focused on two other conserved viral residues (besides the putative active-site Cys) as important to catalysis.…”

Section: Discussionmentioning

confidence: 99%

Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

Bazán

Fletterick

1988

Proc. Natl. Acad. Sci. U.S.A.

397

282

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Proteases that are encoded by animal picornaviruses and plant como-and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, , is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin fl-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses oftrypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases.

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Thioltrypsin

Cited by 45 publications

References 0 publications

Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.

Inactivation of the thiol RTEM-1 β-lactamase by 6-β-bromopenicillanic acid. Identity of the primary active-site nucleophile

Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

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