SUBSITE INTERACTIONS AND RIBONUCLEASE-T1 CATALYSIS - KINETIC STUDIES WITH APGPC AND APGPU

The RNase T1 catalyzed transesterification of ApGpC and ApGpU [to [ApG>p + C] and (ApG>p + U) products, respectively] was studied in steady-state kinetic experiments performed at 25 °C and 0.2 M ionic strength over the pH range 3-9. Values of kcat/Km and Kcat for these substrates were compared, as a function of pH, with those previously determined for GpC and GpU [Osterman, H. L., & Walz, F. G. (1978) Biochemistry 17, 4124], The kinetic properties of ApGpN (N=C or U) substrates differed from those of GpN substrates in the following ways: (1) a dramatic attenuation of the pH dependence of kcai/Km; (2) no discrimination for C or U leaving nucleoside groups; and (3) an opposite pH dependence for /ccat. These results indicate that the adenosine moiety of the trimeric substrates binds with anenzyme subsite and that catalysis can proceed via three parallel reaction paths which are governed by apparent pk values of 5.2 and 7.7 in the enzyme-substrate complex. These paths are characterized by rate constants that are 130, 280, and 1670 which predominate in the acidic, neutral, and basic pH ranges, respectively. Since catalysis of GpN substrates apparently utilizes a single reaction path [characterized by rate constants of 350 and 38 s-1 for GpC and GpU, respectively (Osterman & Walz, 1978)], it is concluded that the mechanism of catalysis for these trimeric and dimeric substrates is different. It is suggested that an active-site carboxylate group serves as a general base catalytic species in all cases except at high pH for ApGpN substrates, where an imidazole residue serves this function. © 1979, American Chemical Society. All rights reserved.