title: Standard free energies for uridylyl group transfer by hexose-1-P uridylyltransferase and UDP-hexose synthase and for the hydrolysis of uridine 5'-phosphoimidazolate.
abstract: The reversible reaction of UDP-glucose with imidazole (Im) to produce uridine 5'-phoshoimidazolate (UMPIm) and glucose-1-P is catalyzed by UDP-hexose synthase, which is the mutant H166G of hexose-1-P uridylyltransferase (EC 2.7.7.12) [Kim, J., Ruzicka, F.J., & Frey, P.A. (1990) Biochemistry 29, 10590-10593]. The availability of UDP-hexose synthase 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 equilibrium constant is 6.4 x 10(-4). The equilibrium constant for the formation of the covalent uridylyl-enzyme intermediate of hexose-1-P uridylyltransferase (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 slightly less favorable than the formation of UMPIm from UDP-glucose and Im. These equilibrium constants, when considered in the light of other data in the literature, allow the standard free energy changes for the hydrolysis of UMPIm and the analogous covalent uridylyl-enzyme intermediate to be calculated. The results show that delta G' degrees (delta G degrees (ph)(7.0)) 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 corresponding values of delta G degrees (ph) (8.5) are -12.6 and -9.9 kcal mol(-1), respectively. It is concluded that noncovalent binding interactions 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 intermediate in the reaction of hexose-1-P uridylyltransferase.
authors.affiliation: Institute for Enzyme Research, University of Wisconsin-Madison 53705, USA.
authors.forename: A
authors.initials: A
authors.lastname: Arabshahi
authors.forename: F J
authors.initials: FJ
authors.lastname: Ruzicka
authors.forename: S
authors.initials: S
authors.lastname: Geeganage
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D013816:Thermodynamics; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry*; D014532:Uridine Diphosphate Glucose / Q000737:chemistry
chemical_list: D007093:Imidazoles; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose