Thiol and amino analogues as alternate substrates for glycerokinase from Candida mycoderma.
The kinetic and catalytic mechanism of glycerokinase from Candida mycoderma was examined with thiol and amino analogues of glycerol and with MgAMPPCP, an analogue of MgATP. (S)-1-Aminopropanediol was phosphorylated on nitrogen (Vmax 0.4% that of glycerol) while the R enantiomer was phosphorylated on oxygen (Vmax 0.7% that of glycerol). (S)-1-Mercaptopropanediol was phosphorylated on oxygen (Vmax 3.5% that of glycerol), while the R enantiomer was phosphorylated on sulfur (Vmax 0.001% that of glycerol). The hydroxyl group at C-2 thus orients the substrate in the active site, while that at the carbon remote from phosphorylation enhances both catalysis and binding of the substrate, presumably because of hydrogen-bonding interactions. The kinetic mechanism is random with a high degree of synergistic binding between the substrates, so that the mechanism appears ordered with glycerol adding first but equilibrium ordered with MgATP binding first with the amino analogues.
Knight
W B
WB
Institute for Enzyme Research, University of Wisconsin, Madison 53706.
Cleland
W W
WW
D000255:Adenosine Triphosphate; D000588:Amines; D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005991:Glycerol Kinase / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds
D000588:Amines; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D005991:Glycerol Kinase