1750
aH(k/Kx). This is similar to eq 2 which describes the rates of hydrolysis of Guo, dGuo, and dAdo. (A spontaneous or "water"-catalyzed reaction is not considered in these derivations.)
Acknowledgment. We are grateful to Professor Joseph F. Bunnett for allowing this project to commence in his laboratory (Brown University) and for initial, valuable discussions.
Relaxation Spectra of Ribonuclease. VII. The Interaction of Ribonuclease with Uridine 2', 3'Cyclic Phosphate' Ernesto J. del Rosario2 and Gordon G. Hammes Contributionfrom the Department of Chemistry, Cornell University, Ithaca, New York 14850. Received September 26, 1969 Abstract: Stopped-flow temperature-jump studies were made of the interaction of ribonuclease A with uridine 2',3 '-cyclic phosphate in 0.2 M NaCI. A bimolecular reaction of enzyme and substrate was observed at pH 6.5, 15', with a second-order rate constant of approximately 1 X lo7 M-l sec-1 and a dissociation rate constant of 2 X lo4 sec-'. A second relaxation process was observed at pH 6.0 and 6.5, 15', which could be associated with an isomerization of the enzyme-substrate complex and the corresponding rate constants were determined. The isomerization of "free" ribonuclease was studied as a function of uridine 2',3 '-cyclic phosphate concentration at 25' in the pH range 5.5-7.5. The data suggest a mechanism where both isomeric forms of the enzyme bind the substrate, and an analogous isomerization can also occur with the enzyme-substrate complexes. arlier papers in this series dealt with the interaction of ribonuclease A with cytidine 3 '-phosphate, 3 , 4 cytidine 2',3 '-cyclic p h ~ s p h a t ecytidilyl-3 ,~ ',5 '-cytidine,6 and uridine 3 ' - p h ~ s p h a t e . ~These studies have indicated that the mechanism of interaction of ribonuclease with all of the mentioned substrates involves an initial association between enzyme and substrate followed by a n isomerization or conformational change of the enzyme-substrate complex. Three ionizable groups on the enzyme with approximate pK values of 5.4, 6, and 6.5 have been implicated in the binding mechanism and presumably the catalytic reaction. Moreover, the enzyme itself was found to undergo an isomerization at neutral p H values, with an ionizable group on the enzyme with a pK of approximately 6 being involved in the conformational change.8 The results of these kinetic investigations have been correlated with the known three-dimensional structure of r i b o n u ~ l e a s elo, ~and ~ a mechanism for the action of the enzyme can be postulated in molecular terms.' The work reported here is concerned with stoppedflow temperature-jump studies of the interaction of ribonuclease A with uridine 2',3 '-cyclic phosphate. A relaxation effect was observed at p H 6.5, 15", which is related to the initial association of enzyme and substrate; a second relaxation process is seen at pH 6 and 6.5, 15", which corresponds to an isomerization of the
E
(1) This work was supported by a grant from the National Institutes of Health (GM 13292). (2) Predoctoral Fellow under the University of the PhilippinesCornell University graduate education program. (3) R. E. Cathou and G. G. Hammes, J . Amer. Chem. Soc., 86, 3240 (1964). (4) R. E. Cathou and G. G. Hammes, ibid., 87,4674 (1965). ( 5 ) J. E. Erman and G. G. Hammes, ibid., 88, 5607 (1966). (6) J. E. Erman and G. G. Hammes, ibid., 88, 5614 (1966). (7) G. G. Hammes and F. G. Walz, Jr., ibid., 91, 7179 (1969). (8) T. C. French and G. G. Hammes, ibid., 87,4669 (1965). (9) G. Icartha, J. Bello, and D. Harker, Nature, 213,862 (1967). (IO) H. W. Wycoff, I
