A synthetic 70-amino acid residue analog of ribonuclease S-protein with enzymic activity.

Abstract

A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.