Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli.
The role of lysine 153 in the action of UDP-galactose 4-epimerase from Escherichia coli has been investigated by site specific mutagenesis and kinetic and spectrophotometric analysis of the mutant enzymes. The crystal structure of UDP-galactose 4-epimerase shows that the binding of NAD+ to the coenzyme site includes the hydrogen bonded interaction of the epsilon-ammonium group of lysine 153 with the 2'- and 3'-hydroxyl groups of the nicotinamide riboside. Mutation of this residue to methionine or alanine decreases the catalytic activity of the enzyme by a factor of more than 10(3). The NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP. NAD+ associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride. However, although the mutant proteins bind UMP very well, the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP. The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site; however, the purified mutants K153M and K153A contain very little NADH. We conclude that lysine 153 plays an important role in increasing the chemical reactivity of enzyme-bound NAD+ in the uridine nucleotide-dependent conformational change associated with reductive inactivation and the catalytic activity of UDP-galactose 4-epimerase.
Swanson
B A
BA
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
Frey
P A
PA
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*; D014542:Uridine Monophosphate / Q000378:metabolism
D011994:Recombinant Proteins; D009243:NAD; D014542:Uridine Monophosphate; D014534:UDPglucose 4-Epimerase; D008239:Lysine