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 UD P-glucose with galactose l-phosphate (Gal-1-P) to form UDP-galactose a nd glucose 1-phosphate (Glc-1-P) through a double displacement mechani sm, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen b onding with the phosphoryl oxygens of the moiety, which is bonded to H is 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. co li variant Q168R has been overexpressed and purified. In preparation f or kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme, At 4 degre es C, the first-order rate constant for uridylylation by UDP-glucose i s 281 +/- 18 s(-1), and for deuridylylation it is 226 +/- 10 s(-1) wit h Glc-1-P and 166 +/- 10 s(-1) with Gal-1-P. Inasmuch as the overall t urnover number at 4 degrees C is 62 s(-1), the covalent intermediate i s kinetically competent. The variant Q168R is uridylylated by UDP-gluc ose to the extent of about 65% of the potential active sites. Uridylyl ation reactions of Q168R with UDP-glucose proceed with maximum first-o rder 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 uridylyl ation of Q168R is slightly higher than for the wild-type enzyme, and f or deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times f aster 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 o f 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 uridyly l-enzyme by 1.2 kcal mol(-1) in comparison to the wild-type enzyme. Th ese results show that the low activity of Q168R is not due to overstab ilization of the intermediate or to the absence of structural metal io ns. Instead, the main defect is very slow uridylylation and deuridylat ion.