The steady-state kinetics of the trypsin-catalyzed hythe trypsin-catalyzed hydrolysis of p-nitrophenyl acedrolysis of the methyl, benzyl, and p-nitrophenyl esters tatea; trans-cinnamoyl-trypsin is seen in the trypsinwere determined at catalyzed hydrolysis of N-trans-cinnam~ylimidazole~;of a-N-benzyloxycarbonyl-L-lysine pH 5.8, using Lineweaver-Burk plots of single experia fast stoichiometric release of p-nitrophenol occurs in ments. The kinetic comparison shown in Table I the trypsin-catalyzed hydrolysis of p-nitrophenyl a-Nindicates that the catalytic rate constants of the three benzyloxycarbonyl-L-lysinate hydrochlorides and the esters are identical within h 6 % , whereas the Km(app) kinetics of the presteady-state and steady-state reacvalues are markedly different. These data are quite tions in this system are satisfactorily described by eq. 1 similar to data on the a-chymotrypsin-catalyzed hyinvolving an acyl-trypsin intermediate.6 drolysis of the ethyl, methyl, and p-nitrophenyl esters of N-acetyl-L-tryptophan.6 Like the chymotrypsin data, the trypsin data are inconsistent with the nucleoHowever, a recent report presents data which tend philic character of trypsin reactions since methyl, to disprove the compulsory formation of an acylbenzyl, and p-nitrophenyl esters have relative rates of enzyme intermediate in the trypsin-catalyzed hydrolysis 1, 1.1, and 50 toward the nucleophile hydroxide i0n.~310 of a-N-benzoyl-L-arginine ethyl ester.’ Moreover, On the other hand, these data are completely consistent the evidence enumerated above may not be general if with the three-step mechanism (eq. 1) for all substrates, one postulates that: (1) the slowly reacting acetyl involving the rate-determining decomposition ( k 3 ) and cinnamoyl derivatives do not necessarily proceed of a common intermediate, a-N-benzyl oxycarbonyl-Lvia the same pathway as the fast arginine and lysine lysyl-trypsin, kz values which reflect the reactivity of derivatives; (2) “activated” p-nitrophenyl esters do these esters toward nucleophiles, and closely similar not necessarily proceed by the same pathway as the K , values. The slight trend in kcat values (kNp” > “nonactivated” methyl or ethyl esters and the amide; kBE = k M E )is in the expected nucleophilic order and and (3) the small spread of reactivities expected in the indicates that deacylation is not completely rate conseries of esters of argument 1 above casts doubt on the trolling; the Km(app) values are also in the expected validity of the argument. Therefore experiments were order (KmNPE
