Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-Variant: Insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose l-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme, At 4 degrees C, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s(-1), and for deuridylylation it is 226 +/- 10 s(-1) with Glc-1-P and 166 +/- 10 s(-1) with Gal-1-P. Inasmuch as the overall turnover number at 4 degrees C is 62 s(-1), the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-4) s(-1) and 4.2 x 10(-4) s(-1) at 4 and 27 degrees C, respectively. In experiments with uridylyl-Q168R and glucose-l-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-4) s(-1) and 1.68 x 10(-3) s(-1) at 4 and 27 degrees C, respectively. The value of K-m for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol(-1) in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.