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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kim
J
J
Institute for Enzyme Research, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705.
Ruzicka
F
F
Frey
P A
PA
D005958:Glucosephosphates / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism; D008956:Models, Chemical; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism
D005958:Glucosephosphates; D007093:Imidazoles; C066970:uridine 5'-(phosphoimidazolate); C029899:imidazole; C031590:glucose-1-phosphate; D014542:Uridine Monophosphate; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose