Chymotrypsin Catalysis in the Presence of Formaldehyde. The pH-Dependence of Ester Hydrolysis

1 The maximum steady state rate (kcat) of the α-chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester at 200 C in 0.15 M KCI is 136 sec−1 and is controlled by a group with pK′ 6.6. From a kcat/k′m versus pH plot, two groups with pK′ 6.6 and 8.3 are seen. In 3.1 M formaldehyde, kcat is 16.3 sec−1 with the rate controlled by a group with pK′ 6.6 and the kcat/K′m versus pH profile is bell-shaped with pK values of 6.4 and 8.4. 2 The active site concentration of α-chymotrypsin in water and in formaldehyde is identical. 3 The modification of chymotrypsin activity by formaldehyde is complete in less than 5 sec and is completely reversible. 4 Diisopropylphosphofluoridate completely inhibits α-chymotrypsin in the presence of formaldehyde although at a slower rate. There is little or no inhibition with L-1-tosylamido-2-phenylethyl chloromethyl ketone. 5 These results lead to the conclusion that the active site has been modified with retention of catalytic activity. From a consideration of the reactivity of formaldehyde with the functional groups of the enzyme, it is suggested that the modification is the result of N-hydroxymethylation of the imidazole group(s) of α-chymotrypsin.