Relaxation kinetics of ribonuclease T1 binding with guanosine and 3'-GMP.
Temperature-jump relaxation kinetic studies were undertaken at 25 degrees C with ribonuclease T1 (RNase T1) alone and in the presence of guanosine (Guo) and 3'-guanylic acid (3'-GMP). No relaxations were observed in the absence of ligands and only one process was observed in their presence which reflected a simple on-off reaction in both cases. Apparent association rate constants, k(on), and dissociation rate constants, k(off), were evaluated at several pH values and their ratios, k(on)/k(off), were contrasted with independently determined values of the equilibrium association constant, Ka(eq). The value of k(on)/k(off) for Guo was significantly greater than Ka(eq), whereas Ka(eq) was significantly greater than k(on)/k(off) for 3'-GMP. The simplest interpretation of the result for Guo is that free RNase T1 undergoes a relatively slow undetected isomerization and Guo can bind only with one isomer. 3'-GMP can be considered to bind with the same preference, but in this case the initial enzyme complex undergoes a relatively slow undetected isomerization. These results are consistent with a recent NMR study which suggested that RNase T1 binding with Guo and 3'-GMP are coupled to slow exchange processes in a ligand dependent manner (Shimada, I. and Inagaki, F. (1990) Biochemistry 29, 757-764). It is tentatively concluded that binding of Guo and 3'-GMP at the active site of RNase T1 is limited to a sub-population of conformers involving the base-recognition site and that the phosphomonoester group of the nucleotide can engage in additional conformationally linked interactions at the adjacent catalytic site.
Walz
F G
FG
Department of Chemistry, Kent State University, OH 44242.
D006151:Guanosine / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism*
D006151:Guanosine; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1