An allosteric model for ribonuclease.

Abstract

Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2': 3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve ofvelocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature.

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

“…At relatively low concentrations of this type of substrate, in an aqueous environment, the only significant route is that of hydrolysis, and a hyperbolic substrate concentration dependence is usually obtained. However, working at high substrate concentrations, Walker et al (4,5) observed that at pH 7.0, the dependence of the rate of hydrolysis with substrate concentration shows a nonhyperbolic behavior: there is a drop at about 25 mM to 30 mM CϾp 1 followed by a second rise and then a gradual decline, which they interpreted by an allosteric model in which there is a substrate-dependent change in the equilibrium between three pre-existing enzyme conformations, one of which inactive. Later, Piccoli and d'Alessio (6) also showed deviations from hyperbolic kinetics for RNase A with CϾp, but only at a low substrate concentration.…”

Maintenance of the monomeric structure of glucokinase under reacting conditions

“…They are ribonucleotide triphosphate reductase (26-28), pancreatic ribonuclease (29)(30)(31), and wheat hexokinase Ll (32). In all these cases there are evidences that the enzymes are single polypeptide chains, but unequivocal proof that they do not oligomerize during the assay has not been reported.…”

The nature of the allosteric interactions of ribonuclease and its ligands

“…Previous studies (Walker et al, 1975(Walker et al, , 1976) have demonstrated that conditions that favour the binding of ligands to the allosteric sites (e.g. increased concentrations of the substrate or its analogues) favour a shift in the equilibrium towards form F of the enzyme.…”

“…Activity was measured at pH 7.13 and 10.3, unless otherwise specified, by using the pH-stat assay described by Walker et al (1975 the pH of the assay system is increased, [Sm] remains constant below pH 8.5 and above pH 10, but increases between these values, reflecting the titration curve for lysine side chains (see Table 1). The slope of the Vol.…”

“…A model for RNAase, activity has been presented (Walker et al, 1975) in which the enzyme exists in two catalytically active forms, E and F. The conformation transition, E=F, is considered to be induced by the binding of a large number of molecules of substrate (or its analogues) to a set of allosteric sites on the enzyme (Walker et al, 1976). The present paper investigates the nature of these allosteric sites.…”