Affinity Labeling of the Active Site of Staphylococcal Nuclease: REACTIONS WITH BROMOACETYLATED SUBSTRATE ANALOGUES

Abstract

Abstract The reaction of staphylococcal nuclease with stoichiometric amounts of 3'-(N-14C-bromoacetyl-p-aminophenylphosphoryl)-deoxythymidine 5'-phosphate, an alkylating reagent that is a derivative of a strong competitive inhibitor, deoxythymidine 3',5'-diphosphate, results in inactivation of enzymatic activity. No reaction occurs in the presence of the competitive inhibitor, or when Ca++, which is required for binding of substrates and inhibitors, is omitted from the reaction mixture. The modified enzyme derivative was readily purified and separated from unreacted enzyme since it did not adsorb to a column containing Sepharose to which the competitive inhibitor, deoxythymidine 3'-p-aminophenylphosphoryl 5'-phosphate, was covalently bound. The radioactivity of this protein derivative was accounted for by N-carboxymethyllysine 48 (36%) N-carboxymethyllysine 49 (40%), and O-carboxymethyltyrosine 115 (15%) in the primary structure of the protein. Only one of these residues is alkylated in a given protein molecule. These residues are presumed to be located in the region of the tertiary structure of the enzyme which affords specific affinity for substrates and inhibitors, but which does not determine the hydrolytic rate constants. Staphylococcal nuclease is also inactivated, stoichiometrically, in the presence of Ca++, by the 14C-bromoacetyl derivative of the poorly hydrolyzed substrate, deoxythymidine 5'-p-aminophenylphosphate. This reagent selectively alkylates tyrosine 85, which is thought to occupy a position near the hydrolytic portion of the active site. The reaction occurs either by alkylation followed by hydrolysis, or by both processes occurring in a concerted fashion, possibly through the initial formation of a phosphorylated enzyme-substrate intermediate. The enzyme derivative modified by alkylation at tyrosine 85 is adsorbed to the specific nucleaseinhibitor Sepharose column, indicating that it possesses residual binding function. The derivative is catalytically active, although its affinity for DNA and synthetic substrates is severely depressed. Reaction of staphylococcal nuclease with the bromoacetyl derivative of deoxythymidine 3'-p-aminophenylphosphate, a weak inhibitor of the enzyme, results in considerable nonspecific and random alkylation. Reaction nevertheless occurs selectively with a residue important for catalysis (lysine 24). Although the amount of this specific reaction is small compared to the total reaction with the protein, as judged by incorporation of 14C, no other single residue appears to be as highly labeled.