Chemical Modifications and Kinetic Study of Ribonuclease Sa Active Site

Chemical modification experiments and kinetic measurements have been carried out to identify the active site components of guanylspecific ribonuclease Sa (E. C. 3.1.4.8.) from Streptomyces aureofaciens. Modification experiments with phenylglyoxal and diketene showed that neither arginine nor lysine residues, which could bind the negatively charged phosphate group of the substrate, belonged to the active site of the enzyme. The pH dependence of the kinetic constants k̂ ,, and Km in the hydrolysis of Guo-P-Cyd and Guo-P-Cyd-P with ribonuclease Sa indicated that a histidine residue could have the above mentioned function. Remarkable reduction (87 per cent) of the enzyme activity after modification with tetranitromethane was found. With these properties ribonuclease Sa considerably differs from ribonuclease Ti.