Galactose-1-phosphate uridylyltransferase: identification of histidine-164 and histidine-166 as critical residues by site-directed mutagenesis.
Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the compulsory formation of a uridylyl enzyme intermediate. The uridylyl group is covalently bonded to the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzyme from E. coli, humans, Saccharomyces, and Streptomyces.
Field
T L
TL
Institute for Enzyme Research, Graduate School, University of Wisconsin--Madison, Madison 53705.
Reznikoff
W S
WS
Frey
P A
PA
D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005809:Genes, Regulator; D006639:Histidine / Q000378:metabolism*; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D014169:Transformation, Bacterial; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D006639:Histidine; D001216:Asparagine; D009713:Nucleotidyltransferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase