Alkylation of a histidine residue at the active site of bovine pancreatic deoxyribonuclease.

In the study of reactions designed to identify the amino acid residues most directly involved in the catalytic function of pancreatic deoxyribonuclease, the enzyme was tist treated with iodoacetate under conditions similar to those which are effective in alkylating essential histidine residues in pancreatic ribonuclease; no inactivation of DNase occurred although some reagent was incorporated. DNase is an enzyme which requires divalent cations to be present with the substrate; when the iodoacetate reaction was tried in the presence of 0.1 M Mn*, DNase was inactivated at pH 7.2 in Tris-chloride buffer and the loss of activity paralleled the formation of 1 residue of 3-carboxymethylhistidine per molecule. The variation of the rate of inactivation with Mn++ concentration indicated that alkylation by iodoacetate is facilitated by the binding of 1 Mn+f per molecule of enzyme. Substitution of Mn* by 0.1 mu Cu* greatly increases the rate of alkylation; the half-life of the DNase activity is 5 min at 25O and the only substituted amino acid formed is 3-carboxymethylhistidine. A 29-residue peptide containing the modified histidine residue has been isolated in 90% yield after tryptic hydrolysis of the carboxymethylated DNase. The phenomena of substrate hydrolysis and carboxymethylation at the active site are thus both metal-dependent. The presence of Mn++ or Cu++ probably facilitates the alkylation by increasing the affinity of the active site for the carboxy1 group of iodoacetate. A neutral alkylating agent, iodoacetamide, is without effect. The present results increase the number of enzymes of pancreatic origin but differing specificities which are known to have histidine at the active site.