The role of serine-123 in the activity and specificity of ribonuclease. Reactivation of ribonuclease 1-118 by the synthetic COOH-terminal tetradecapeptide, ribonuclease 111-124, and its O-methylserine and alanine analogs.

Abstract

The COOH-terminal tetradecapeptide of ribonuclease A, Glu-Gly-Asn-Pro-Tyr-Val-Pro-Val-His-Phe-Asp-Ala-Ser-Val, and two analogs, [Ser(Me)-123]-RNase 111-124 and [Ala-123]-RNase 111-124, were synthesized by the solid phase method and were purified to chromatographic and electrophoretic homogeneity. Methods are described for the hydrolysis and quantitative amino acid analysis of peptides containing O-methylserine. The peptides were combined noncovalently with RNase 1-118 and examined for ability to regenerate enzymatic activity in the presence of the substrates C greater than p, U greater than p, poly(C) poly(U), and poly(AF). The dissociation constants of the peptide-protein complexes, and the Michaelis constants for C greater than p and U greater than p with the reconstituted enzymes were determined. The data were used to test hypotheses, drawn from x-ray crystallographic and other studies, for the role of serine-123 in the binding of substrates by ribonuclease. It was found that Ser-123- and Ala-123-containing peptides were equally active for the hydrolysis step when measured with C greater than p as substrate and for the transphosphorylation step as measured in the assays with poly(C). The serine and alanine analogs were also equally active for the transphosphorylation step when poly AF was the substrate. With U greater than p as substrate the alanine analog was 4 times less active than the serine derivative and with poly U it was 2 times less active. The semisynthetic enzyme composed of RNase 1-118 and [Ala-123]-RNase 111-124, therefore, shows appreciable selectivity for substrates containing cytosine. It was concluded that a hydrogen bond between the hydroxyl of serine-123 and the C4 amino group of cytidine or the C-7 amino group of formycin is not important for substrate binding and catalytic activity. In contrast, the hydrogen bond between the hydroxyl of serine 123 and the C-4 carbonyl oxygen of uridine contributes significantly to substrate binding and catalytic activity. The data with serine-O-methyl ether at position 123 in the tetradecapeptide were less clear because it was difficult to separate steric effects from the contributions of hydrogen bonding. Substrate binding to ribonuclease was rationalized in terms of a binding energy equivalent to a total of two hydrogen bonds per pyrimidine.