STANDARD FREE-ENERGIES FOR URIDYLYL GROUP-TRANSFER BY HEXOSE-1-P URIDYLYLTRANSFERASE AND UDP-HEXOSE SYNTHASE AND FOR THE HYDROLYSIS OF URIDINE 5'-PHOSPHOIMIDAZOLATE

The reversible reaction of UDP-glucose with imidazole (Im) to produce uridine 5'-phosphoimidazolate (UMPIm) and glucose-1-P is catalyzed by UDP-hexose synthase, which is the mutant H166G of hexose-1-P uridylylt ransferase (EC 2.7.7.12) [Kim, J., Ruzicka, F. J., & Frey, P. A. (1990 ) Biochemistry 29. 10590-10593]. The availability of UDP-hexose syntha se allows the equilibrium constant for the reaction UDP-glucose + Im = UMPIm + glucose-1-P to be measured, and it is found to be 2.2 x 10(-2 ) at pH 8.5 and 27 degrees C. At pH 7.0 and 27 degrees C the equilibri um constant is 6.4 x 10(-4) The equilibrium constant for the formation of the covalent uridylyl-enzyme intermediate of hexose-1-P uridylyltr ansferase (E-His(166) + UDP-glucose = E-His(166)-UMP + glucose-1-P) is found to be 1.8 x 10(-4) at pH 7.0 and 25 degrees C, which is slightl y less favorable than the formation of UMPIm from UDP-glucose and Im. These equilibrium constants, when considered in the light of other dat a in the literature, allow the standard free energy changes for the hy drolysis of UMPIm and the analogous covalent uridylyl-enzyme intermedi ate to be calculated. The results show that Delta G'degrees (Delta G d egrees(pH7.0)) for the hydrolyses of UMPIm and E-His(166)-UMP are -14. 7 and -15.4 kcal mol(-1), respectively at pH 7.0. At pH 8.5, the corre sponding values of Delta G degrees(pH8.5) are -12.6 and -9.9 kcal mol( -1), respectively. It is concluded that noncovalent binding interactio ns between the active site and the UMP group of E-His(166)-UMP provide little or no stabilization in the formation of this species as an int ermediate in the reaction of hexose-1-P uridylyltransferase.