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 Escheri chia coli catalyzes the interconversion of UDP-glucose and galactose-1 -P with UDP-galactose and glucose-1-P by a double-displacement mechani sm through a uridylyl-enzyme intermediate, in which the uridine-5'-pho sphoryl group is covalently bonded to N-epsilon of His 166. The point variant H166G displays a UDP-hexose synthase activity, in that it cata lyzes the reaction of uridine 5'-phosphoimidazolide (UMPIm) with gluco se-1-P to form UDP-glucose and imidazole. Inasmuch as the wild-type ur idylyltransferase catalyzes its cognate reaction with ping-pong kineti cs, an intrinsically ordered substrate binding mechanism, the kinetic mechanism of the UDP-hexose synthase activity of H166G became of inter est. The synthase activity follows sequential kinetics [Kim, J., Ruzic ka, F., and Frey, P. A. (1990) Biochemistry 29, 10590-10593]. In this work, product inhibition patterns for the synthase activity of H166G i ndicate random equilibrium binding of substrates. Comparison of the sy nthase activities of the variants H166G and H166A showed that the glyc ine variant is about 340- and 600-fold more active than the alanine va riant in the forward and reverse directions, respectively. The kinetic consequences of varying the amino acid at position 166 were largely k (cat) effects, with more modest K-m effects. Comparison of the synthas e activities of these variants with that of the wild-type enzyme in th e 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 fo r uridylyl group transfer by 2.4 kcal mol(-1), and the presence of two additional hydrogen atoms in the complex H166A-UMPIm increases the ac tivation energy by 6.2 kcal mol(-1). It is concluded that the active s ite is much less tolerant of additional steric bulk in the locus of th e beta-carbon of His 166 than it is of the loss of the beta-carbon. Th e sensitivities to additional steric bulk around other positions of th e His 166-imidazole ring are much less severe, as indicated by the rea ctivities of methylated analogues of UMPIm in the synthase reaction of H166G, Uridine 5'-phospho-N-methylimidazolide is more reactive as a s ynthase substrate than UMPIm, and this is attributed to the positive c harge of the imidazole ring. The fact that the imidazole ring of the w ild-type covalent uridylyl-enzyme retains its proton and is positively charged is supported by the pH-rate profile for hydrolysis of the int ermediate.