RELAXATION SPECTRA OF RIBONUCLEASE .6. INTERACTION OF RIBONUCLEASE WITH URIDINE 3'-MONOPHOSPHATE

Kinetic studies of the interaction of ribonuclease with uridine 3′-monophosphate were performed using the temperature-jump method. Changes in both ultraviolet absorption and pH were measured. A partial reinvestigation of the relaxation spectra of ribonuclease-cytidine 3 ′-monophosphate binding was also carried out. For both nucleotides two relaxation processes are observed which can be attributed to an initial association-dissociation of the enzyme and nucleotide followed by an isomerization of the enzyme-nucleotide complex. These two processes were studied at 25˚ in the pH range 4.5-7.5. The interaction of ribonuclease with both uridine and cytidine 3 ′-nucleotides has qualitatively similar relaxation characteristics as a function of pH. However, the dissociation rate constant for uridine 3′-monophosphate binding is approximately two times that for cytidine 3 ′-monophosphate. A minimal mechanism consistent with all of the data involves three ionizing groups on ribonuclease and two isomeric states of the ribonuclease-3′-nucleotide complex. One of the ionizing groups on ribonuclease is postulated not to interact directly with the 3 ′-nucleotides, but instead reflects a change in conformation associated with the isomerization of the free enzyme and the enzyme-product complex. These results can be correlated with the known three-dimensional structure of the enzyme. © 1969, American Chemical Society. All rights reserved.