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 aden ine and ribose 5-phosphate. Kinetic isotope effects have been used to establish an experimentally based transition-state structure for the n ative enzyme and a V,, mutant [Mentch, F., Parkin, D.W., and Schramm, V.L. (1987) Biochemistry 26, 921-930; Parkin, D.W., Mentch, F., Banks, G.A., Horenstein, B.A., and Schramm, V. L. (1991) Biochemistry 30, 45 86-4594]. The transition states are characterized by weak reaction coo rdinate bonds to Cl' and substantial carbocation character in the ribo se ring. The N9-Cl' bond to the leaving group is nearly broken and the adenine ring is protonated at the transition state. Formycin 5'-phosp hate and other purine nucleoside 5'-phosphate analogues with syn-glyco syl torsion angles bind better than substrate, supporting a syn config uration in the enzyme-substrate complex and presumably in the transiti on state [Giranda, V.L., Berman, H.M., and Schramm, V.L. (1988) Bioche mistry 27, 5813-5818]. Access to a geometric model of the transition s tate permits the analysis of its molecular electrostatic potential sur face as enforced by the enzyme. Comparison of the molecular electrosta tic potential surfaces for AMP, formycin 5'-phosphate, and the transit ion state reveals a striking similarity in the surface charges of form ycin 5'-phosphate and the transition state. The enzyme-stabilized tran sition state for AMP hydrolysis is characterized by new positive elect rostatic potential in the adenine ring as a result of protonation by t he 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 K-m/K-i value of >10(3) for formy cin 5'-phosphate as a transition-state inhibitor of AMP nucleosidase.