Intramolecular reaction of specific substrate as a step in α‐chymotrypsin catalyzed hydrolysis

Ma, Che; Axelrud-Cavadore, Claude; Previero, A.

doi:10.1016/0014-5793(70)80531-2

Cited by 16 publications

(4 citation statements)

References 14 publications

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“…An intermediate between ES and EA in the reaction with 7V-(2-furyl)acryloyl-L-tyrosine methyl ester at low pH has been observed spectrophotometrically (Yu and Viswantha, 1969). The rate of formation of this intermediate is similar to that for oxazolinone formation in the corresponding tryptophan substrate (Coletti-Previero et al, 1970) and of the same magnitude as that estimated for reaction 4 of IV-acetyl-L-phenylalanine p-nitroanilide under similar conditions. Using a combination of relaxation and stopped-flow techniques, Hess (Hess et al, 1970) demonstrated the existence of an intermediate between ES and EA using A-(2-furyl)acryloyl-L-tryptophan amide as substrate.…”

Section: Discussionsupporting

confidence: 52%

“…The formation of the acyl enzyme involves the "charge-relay" system (Blow et al, 1969) consisting of the catalytic triad Ser-195, His-57, Asp-102 (Hunkapiller et al, 1973;Robillard and Shulman, 1972;Brinigar and Chao, 1975). Several studies of chymotrypsin catalysis have suggested the existence of additional intermediates to those shown in (1): investigations have been done using 7V-furylacryloyl substrate derivatives (Hess et al, 1970; Yu and Viswantha, 1969; Coletti-Previero et al, 1970), nitrogen isotope effects (O'Leary and Kluetz, 1972), and the pH dependence of kinetic parameters (Caplow, 1969;Lucas and Caplow, 1972;Lucas et al, 1973;Fersht, 1972a;Fastrez and Fersht, 1973a;Fersht and Requena, 1971;Fersht and Renard;1974).…”

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confidence: 99%

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Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate

Fink¹

1976

Biochemistry

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The reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitroanilide has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvent. Following initiation of the reaction at temperatures as low as -90 degrees C, a series of four reactions prior to the normal rate-limiting step (acylation) was detected spectrophotometrically. Various experimental observations have led to the following interpretation of these reactions. Reaction 1 corresponds to the binding of substrate yielding the initial Michaelis complex. Reactions 2 and 3 are two pH-independent reactions, ascribed to substrate-induced changes in the positions of active-site groups. Reaction 4 is a pH-dependent reaction (pK = 5.9) which involves the imidazole of His-57 but which is not the formation of a tetrahedral intermediate, oxazolinone, or acyl enzyme. The slowest detected step corresponded to the acylation reaction. No evidence for the accumulation of a tetrahedral intermediate was obtained. Spectral, kinetic, and thermodynamic data for these reactions are presented, as is justification for the relevance of these findings to the reaction under physiological conditions. These results demonstrate the utility of subzero temperatures in enzyme mechanism studies, especially with regard to allowing the accumulation of intermediates which may be quite stable at appropriate values of pH and low temperature.

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Section: Discussionsupporting

confidence: 52%

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confidence: 99%

Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate

Fink¹

1976

Biochemistry

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“…Such an oxazoline has actually been isolated from the reaction of furylacryloyl-tryptophan methyl ester with chymotrypsin at low pH. 12…”

Section: F Wudl Bell Laboratoriesmentioning

confidence: 99%

Kinetic isotope effects for the chymotrypsin catalyzed hydrolysis of ethoxyl-18O labeled specific ester substrates

Sawyer¹,

Kirsch²

1975

J. Am. Chem. Soc.

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“…Department of Biochemistry, University of California Berkeley, California 94720 Received October12, 1974 Mechanisms of Grignard Reactions with Ketones. Polar vs.…”

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Mechanisms of Grignard reactions with ketones. Polar vs. single electron transfer pathways

Ashby¹,

Lopp²,

Buhler³

1975

J. Am. Chem. Soc.

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Intramolecular reaction of specific substrate as a step in α‐chymotrypsin catalyzed hydrolysis

Cited by 16 publications

References 14 publications

Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate

Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate

Kinetic isotope effects for the chymotrypsin catalyzed hydrolysis of ethoxyl-18O labeled specific ester substrates

Mechanisms of Grignard reactions with ketones. Polar vs. single electron transfer pathways

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