Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase.
Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.
Gerratana
B
B
Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Cleland
W W
WW
Frey
P A
PA
D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry* / Q000235:genetics; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D013942:Thymine Nucleotides / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014534:UDPglucose 4-Epimerase / Q000737:chemistry; D014994:Xylose / Q000031:analogs & derivatives / Q000737:chemistry
D013942:Thymine Nucleotides; C103522:deoxythymidine diphosphate-glucose; D009243:NAD; D013912:Threonine; D014443:Tyrosine; D014994:Xylose; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D014534:UDPglucose 4-Epimerase; D017875:Phosphotransferases (Phosphomutases); D005947:Glucose; D008239:Lysine