REMODELING HEXOSE-1-PHOSPHATE URIDYLYLTRANSFERASE - MECHANISM-INSPIRED MUTATION INTO A NEW ENZYME, UDP-HEXOSE SYNTHASE

Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166. To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted. To determine whether the function of the deleted imidazole in these mutants could be carried out by the imidazole ring in uridine 5′-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole. The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates. The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose. Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction. The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase. The value of kcat in the direction of UDP-glucose formation is 1.31 ± 0.01 s−1, compared with 350 s−1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 ± 0.4 s−1, compared with 960 s−1 for the wild-type enzyme. Thus, the kinetic “price” that is paid for removing the covalent bond and a methylene group linking the enzyme and the nucleophilic imidazole in the parent enzyme is an approximately 250-fold decrease in turnover number. The mutant H164G has no detectable catalytic activities. We conclude that the imidazole ring of H166 is the nucleophilic catalytic group of hexose-1-P uridylyltransferase. The mutant protein H166G is a new enzyme that catalyzes a new reaction by a kinetic mechanism different from that of its parent enzyme. We suggest the name UDP-hexose synthase for this new enzyme. © 1990, American Chemical Society. All rights reserved.