Effect of Alkylguanidines on the Inactivation of Trypsin by Alkylation and Phosphorylation

Abstract

Abstract It has previously been shown that methylguanidine increases, by a factor of 7, the rate of the trypsin-catalyzed hydrolysis of acetylglycine ethyl ester, whereas n-butylguanidine inhibits the reaction. These effects have been postulated to be due to the binding of the effectors at the recognition site of the enzyme which results in an induced conformational change of the enzyme. In this study such induced effects were analyzed in terms of changes in chemical reactivity of functional groups in the active site of the enzyme. A comparison has been made of the effects of methylguanidine and n-butylguanidine with respect to the alkylation of histidyl residues by α-halo-acetic acid derivatives and with respect to the phosphorylation of the active seryl residue by diisopropyl fluorophosphate. The presence of methylguanidine increased the rate of the inactivation by iodoacetamide by a factor of 6 compared to the rate in the absence of the alkylguanidine, whereas n-butylguanidine decreased the rate. Data were obtained which suggested that the carboxamidomethylation of the 3-N atom of an imidazole of histidine caused such inactivation. The observed 7-fold increase in catalytic rate coupled with the concomitant 6-fold increase in the rate of alkylation strongly suggests that the binding of methylguanidine to the recognition site of the enzyme alters the reactivity of an imidazole group (groups). The phosphorylation reaction by diisopropyl fluorophosphate was completely prevented by both methyl- and n-butylguanidine. A generalized approach for studying the effects of conformational change of an enzyme molecule in terms of changes in the chemical reactivity of functional groups in the active site is proposed.