Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Abstract

The pH, ionic strength and solvent deuterium isotope dependence of the steady-state kinetics of the RNase A-catalyzed hydrolysis of 2'',3''-cCMP was investigated by using, primarily, the technique of flow microcalorimetry to monitor the kinetics. The pH dependence of the Michaelis-Menten parameters was analyzed by assuming the participation of His-12 and -119 of the enzyme and a third ionizing group, postulated to be on the pyrimidine ring of the substrate, to determine the pH-independent rate constant, kc, and Km. The reported pH analysis, together with existing NMR data and chemical modification studies, allows an assignment of the functional roles of His-12 and -119 as being those of general acid and general base catalytic residues, respectively. At high pH, the apparent Km value increases to unity. This drop in affinity between the enzyme and the substrate at high pH indicates that the substrate binds to the enzyme primarily through an electrostatic interaction with the active-site histidine residues, particularly His-12. The apparent absence of an interction with the riboside portion of the substrate may be due to the fact that the substrate exists in a syn conformation about its glycosidic bond and thus cannot interact optimally with the enzyme''s binding pocket. This will result in a relative destabilization of the enzyme-substrate complex, which can then be relieved upon the formation of the transition state. The ionic strength dependence of RNase activity is primarily a result of its effect on the pKa of the histidine residues and a concomitant change in the value of Km.