Electrostatic potential surface analysis of the transition state for AMP nucleosidase and for formycin 5'-phosphate, a transition-state inhibitor.

AMP nucleosidase hydrolyzes the N-glycosidic bond of AMP to yield adenine and ribose 5-phosphate. Kinetic isotope effects have been used to establish an experimentally based transition-state structure for the native enzyme and a Vmax mutant [Mentch, F., Parkin, D. W., & Schramm, V. L. (1987) Biochemistry 26, 921-930; Parkin, D. W., Mentch, F., Banks, G. A., Horenstein, B. A., & Schramm, V. L. (1991) Biochemistry 30, 4586-4594]. The transition states are characterized by weak reaction coordinate bonds to C1' and substantial carbocation character in the ribose ring. The N9-C1' bond to the leaving group is nearly broken and the adenine ring is protonated at the transition state. Formycin 5'-phosphate and other purine nucleoside 5'-phosphate analogues with syn-glycosyl torsion angles bind better than substrate, supporting a syn configuration in the enzyme-substrate complex and presumably in the transition state [Giranda, V. L., Berman, H. M., & Schramm, V. L. (1988) Biochemistry 27, 5813-5818]. Access to a geometric model of the transition state permits the analysis of its molecular electrostatic potential surface as enforced by the enzyme. Comparison of the molecular electrostatic potential surfaces for AMP, formycin 5'-phosphate, and the transition state reveals a striking similarity in the surface charges of formycin 5'-phosphate and the transition state. The enzyme-stabilized transition state for AMP hydrolysis is characterized by new positive electrostatic potential in the adenine ring as a result of protonation by the enzyme. This is closely matched by the protonated pyrazole ring of formycin 5'-phosphate. The molecular electrostatic potential surfaces of formycin 5'-phosphate and the transition state for AMP are similar and are likely to be a factor in the Km/Ki value of > 10(3) for formycin 5'-phosphate as a transition-state inhibitor of AMP nucleosidase.