Rate-limiting reaction in papain action as derived from the reaction of the enzyme with chloroacetic acid

Chloroacetic acid reacts with the essential thiol group of papain (EC 3.4.4.10) and inactivates the enzyme. The rate of inactivation, k, at pH 6.0, 25°, was determined in the absence and presence of the substrates benzoylarginine ethyl ester (BAEE) and benzoylglycine ethyl ester (BGEE). The rate was found to be independent of BAEE concentration and to decrease with increasing BGEE concentration. Extrapolation to infinite BGEE concentration yielded k = o. These results indicate that (a) acylation of the thiol group is the rate-limiting step in BAEE hydrolysis, (b) deacylation is the rate-limiting step in BGEE hydrolysis. The thiol group of papain is 30 000 times more reactive towards chloroacetic acid than is the thiol group of cystine at pH 6. ¿max values were determined for four benzoylarginine derivatives, the methyl, ethyl and propyl esters and the amide. These values were found to differ by no more than a factor of three. It is pointed out that caution should be exerted in interpreting ¿max data in terms of rate-limiting steps.