Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis

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Abstract
Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3′-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5′-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5′-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5′-AMP effectively, suggests the involvement of histidine in catalysis.