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Kinetic mechanism of UDP-hexose synthase, a point variant of hexose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate (galactose-1-P) uridylyltransferase from Escherichia coli catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism through a uridylyl-enzyme intermediate, in which the uridine-5'-phosphoryl group is covalently bonded to Nepsilon of His 166. The point variant H166G displays a UDP-hexose synthase activity, in that it catalyzes the reaction of uridine 5'-phosphoimidazolide (UMPIm) with glucose-1-P to form UDP-glucose and imidazole. Inasmuch as the wild-type uridylyltransferase catalyzes its cognate reaction with ping-pong kinetics, an intrinsically ordered substrate binding mechanism, the kinetic mechanism of the UDP-hexose synthase activity of H166G became of interest. The synthase activity follows sequential kinetics [Kim, J., Ruzicka, F., and Frey, P. A. (1990) Biochemistry 29, 10590-10593]. In this work, product inhibition patterns for the synthase activity of H166G indicate random equilibrium binding of substrates. Comparison of the synthase activities of the variants H166G and H166A showed that the glycine variant is about 340- and 600-fold more active than the alanine variant in the forward and reverse directions, respectively. The kinetic consequences of varying the amino acid at position 166 were largely kcat effects, with more modest Km effects. Comparison of the synthase activities of these variants with that of the wild-type enzyme in the production of glucose-1-P showed that the loss of the beta-carbon of His 166 in the complex H166G-UMPIm increases the activation energy for uridylyl group transfer by 2.4 kcal mol-1, and the presence of two additional hydrogen atoms in the complex H166A-UMPIm increases the activation energy by 6.2 kcal mol-1. It is concluded that the active site is much less tolerant of additional steric bulk in the locus of the beta-carbon of His 166 than it is of the loss of the beta-carbon. The sensitivities to additional steric bulk around other positions of the His 166-imidazole ring are much less severe, as indicated by the reactivities of methylated analogues of UMPIm in the synthase reaction of H166G. Uridine 5'-phospho-N-methylimidazolide is more reactive as a synthase substrate than UMPIm, and this is attributed to the positive charge of the imidazole ring. The fact that the imidazole ring of the wild-type covalent uridylyl-enzyme retains its proton and is positively charged is supported by the pH-rate profile for hydrolysis of the intermediate.