<b>Co-operative Effects of Functional Groups in Peptides. I. Aspartyl-serine Derivatives</b>

Bernhard, Sidney A.; Berger, Arieh; Carter, John H.; Katchalski, Ephraim; Sela, Michael; Shalitin, Yechiel

doi:10.1021/ja00871a030

Cited by 108 publications

(38 citation statements)

References 2 publications

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“…3 Lower), derived from the thiolester in the fast step, ka. This is consistent with the hydrolysis of aspartyl esters, in which the imide formation is faster than its hydrolysis (14 CONCLUSIONS AND SPECULATIONS (i) We have presented circumstantial evidence for an active-site thiolester in a2M. Because we are unable to eliminate the possibility that the alkylamine-exposed thiol is a noncovalent buried group, direct chemical evidence for a thiolester is needed.…”

Section: Methodssupporting

confidence: 62%

“…If the putative thiolester was hydrolyzed during protein denaturation, the mechanism must include intramolecular assistance to account for the rate ofconversion-namely, the calculated pseudo-firstorder rate constant for a simple thiolester hydrolysis at 25°C and pH 8.0 is -5 X 10-6 min-' (13), whereas that for the deactivation of a2M can be estimated to be >1 X 10-2 min1. This magnitude of rate enhancement is similar to that for the hydrolysis of aspartyl peptide 1-esters (14). However, acetylimidazole (an alternative model for the glutamyl activation) is hydrolyzed without anchimeric assistance at a rate similar to that for the a2M active site (15).…”

Section: Methodssupporting

confidence: 57%

“…3 Lower. An intermediate analogous to B was found for the hydrolysis ofl3-aspartyl esters (14) and for the a --y amide shift in glutamyl peptides (16,17). Hydrolysis of B favored the formation of y amides.…”

Section: Methodsmentioning

confidence: 83%

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Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

Howard¹

1981

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

128

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The reaction ofmethylamine with a2-macroglobulin (a2M) and iodo[2-3H]acetic acid, a tryptic peptide was isolated that contained both labels in the same ratio as in the original protein. From the chymotryptic digest of the tryptic peptide, a single radiolabeled peptide was isolated. The amino acid sequence of the chymotryptic peptide was the same as that previously reported to include y-glutamylmethylamide. This is circumstantial evidence for a thiolester between the cysteine and a glutamic acid located three residues away in the primary sequence. A reaction mechanism involving a pyroglutamyl intermediate derived from the thiolester is suggested to explain the autolysis. Kinetic analysis of the autolysis reaction is consistent with this intermediate and mechanism.The human plasma proteins a2-macroglobulin (a2M), complement component C-3, and complement component C-4 have a unique reactivity with nucleophiles, a property that can lead to complete inactivation of the protein (1-5). We have shown that, when methylamine is incubated with one ofthese proteins, a specific glutamic residue is irreversibly and covalently modified as y-glutamylmethylamide (1-3, 5). For a2M, the amino acid sequence ofa 56-residue glycopeptide containing the modified residue has been reported (2).A second property common to these proteins is the lability of a single peptide bond in the partially denatured protein (3,(5)(6)(7)(8)(9). Upon incubation of the protein at 80°C in buffer alone, or at lower temperatures in denaturing buffers, the protein is specifically fragmented-e.g., the a2M subunit (Mr 185,000) is cleaved into peptides ofMr 115,000 and 65,000 (fragments I and II), the a-subunit of C-3 is cleaved into peptides of Mr 80,000 and 55,000, and the a-subunit of C-4 is cleaved into peptides of Mr 60,000 and 45,000. The denaturation-dependent peptide bond hydrolysis can be prevented by prior reaction of the protein with nucleophiles. For C-3 and C-4, which are composed ofmore than one type ofsubunit, the methylamine-reactive site and the peptide-hydrolysis site are in the same subunit (3, 5). Recently, we (8) reported for a2M that the site offragmentation is the peptide bond on the amino-terminal side of the reactive glutamic residue. The cleavage proceeds with the concerted formation ofan amino-terminal pyroglutamic acid for the newly formed peptide fragment, fragment II (the carboxyl-terminal third of the original a2M subunit). Because nucleophilic substitution and peptide bond scission occur at the same glutamic residue in a2M, we (2, 8) have concluded that both phenomena are the consequence of a single highly activated residue. The similarity of both reactions in a2M, C-3, and C-4 led us (1,2,8) to suggest that the active sites in three proteins should have sequence homology and a common mechanism of activation. The sequence homology in the active site of a2M (1, 2) and C-3 (4) has been confirmed.Although we (1-3) have hypothesized that the carboxyl group could be activated by either an oxyester or a thiolester, the nature...

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Section: Methodssupporting

confidence: 62%

Section: Methodssupporting

confidence: 57%

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Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

Howard¹

1981

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

128

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“…This Asn deamidation in proteins and peptides in aqueous solution can proceed at a much higher rate than is observed for hydrolysis of an amide linkage of a peptide bond [70,72,73], and the increased rate suggests an intramolecular reaction [74,75]. Some deamidation in peptides and proteins is known to occur through intramolecular attack of the carboxy-peptide-nitrogen on the side-chain γ-carbonyl carbon, resulting in the formation of a succinimide ring [72,76].…”

Section: Asparagine Residuesmentioning

confidence: 99%

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

277

175

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Now that enzymes are available that are stable above 100 mC it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in

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“…This increased rate of hydrolysis is similar to the greatly accelerated rate of hydrolysis of esters having amino, carbonyl or hydroxyl as neighbouring groups (Bernhard et al 1962;Shalitin & Bernhard, 1964a,b).…”

Section: Discussionmentioning

confidence: 62%

Interaction of N-alkyl-N-nitrosourethanes with thiols

Schoental

Rive

1965

Biochemical Journal

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1. The interaction at room temperature of thiols (cysteine and certain of its derivatives, and glutathione) with N-alkyl-N-nitrosourethanes and with diazomethane gave complicated mixtures of products. 2. S-Methylcysteine and S-ethoxycarbonylcysteine, the main products of the reaction between cysteine and N-methyl-N-nitrosourethane, were isolated and unequivocally identified. Paper-chromatographic and other evidence was used for the identification of several other components of the mixtures of products obtained. 3. S-Methyl derivatives and their methyl esters were the common products formed from the thiol compounds with both N-methyl-N-nitrosourethane and with diazomethane. 4. The esters were unstable and hydrolysed readily. 5. S-Ethoxycarbonyl derivatives, the primary products of the reaction of the thiols with N-alkyl-N-nitrosourethanes, isomerized to the respective N-ethoxycarbonyl derivatives when the pH increased above 7.0; the migration of the ethoxycarbonyl group from S to N was particularly easy with cysteine, probably owing to the spatial proximity of the amino group. 6. It is suggested that the non-enzymic reactions in vitro of thiols with both N-methyl-N-nitrosourethane and diazomethane may represent models for events in vivo and might help in the studies of the carcinogenic process.

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Co-operative Effects of Functional Groups in Peptides. I. Aspartyl-serine Derivatives

Cited by 108 publications

References 2 publications

Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

The denaturation and degradation of stable enzymes at high temperatures

Interaction of N-alkyl-N-nitrosourethanes with thiols

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