The kinetics of hydrolysis of derivatives of arginine, homoarginine and ornithine by trypsin

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Abstract
The synthesis of sev-eral new substrates for trypsin is described. The occurrence of activation by the substrate during the trypsin-catalysed hydrolysis of [alpha]-N-toluene-p-sulphonyl-L-arginine methyl ester has been confirmed. The kinetics of the hydrolysis of this and the corresponding ethyl, n-propyl and cyclohexyl esters are statistically indistinguishable. It is concluded that K3 (app.) refers to the deacylation of ([alpha]a-N-toluene-p-sulphonyl-L-arginyl)-trypsin. Esters of [alpha]-N-toluene-p- sulphonyl-L-homo-arginine are substrates for trypsin, although Km(app.) is considerably higher than for the corresponding arginine compound*. The identity of k3 (app.) for the methyl, ethyl and n-propyl esters indicates that the deacylation of ([alpha]-N-toluene-p-sul-phonyl-L-homoarginyl)trypsin is rate-determining. The difference between the values of ks (app.) for the arginine and homoarginine derivatives is mainly due to a difference in entropy of activation. The variation of k3 (app.) with pH for N-toluene-p-sulphonyl-L-arginine methyl ester suggests that a group with pK(app.) 7.85 [plus or minus] 0.05 must be dissociated for deacylation of the intermediate acylated enzyme to proceed. With [alpha]-N-toluene-p-sulphonyl-L-homoarginine methyl ester, the pK(app.) value is 6.93 [plus or minus]0.04. Trypsin catalyses the hydrolysis of [alpha]-N-toluene-p-sulphonylornithine methyl ester at pH 7[center dot]0, but the reaction is not completely stereospecific.

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