Abstract
The apparent first-order rate constant (kobs) for the deacylation of α-benzamido-trans-cinnamoyl-chymotrypsin, determined by direct observation at 310 nm, pH 7.9 (kobs= 0.102 s−1; K. Brockelhurst and K. Williamson [1967] Chem. Commun., 666), differs from kcat determined under the same conditions with [S]0≫ [E]0 (kcat= 0.033 s−1). This result is contrary to other evidence indicating that kcat is a measure of the deacylation rate constant. The discrepancy has been shown to be due to the presence in commercial samples of α-chymotrypsin of impurities which catalyse the deacylation reaction under the conditions used for the determination of kobs ([E]0 > [S]0). The impurities, which may be removed from enzyme solutions by gel filtration on Sephadex G-25 or G-50, have been shown to be autolysis products (presumably peptides) of α-chymotrypsin. Catalysis of deacylation results from attack of the fre α-amino groups of these peptides on the acyl-enzyme, to give N-α-benzamido-trans-cinnamoyl-peptides. The consequences of these findings must be considered in interpreting any experiment in which deacylation is observed directly at high enzyme concentration, with [E]0 > [S]0. Particularly susceptible to error are experiments designed to determine the effect of pH on the deacylation rate constant, and literature results of some such experiments are critically assessed.
Published Version
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