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Abstract
Glycerol kinase (E.C. 2.7.1.30, ATP: glycerol-3-phosphotransferase) catalyzes the phosphorylation of glycerol, dihydroxyacetone, and certain structural analogues of glycerol by ATP. The reaction is highly stereospecific. Chapter One examines the limitations in substrate specificity of glycerol kinase and the differences in substrate specificity with enzyme source. A survey of 66 structural analogues of glycerol has defined qualitatively the structural characteristics necessary for acceptance as a substrate for this enzyme. These studies indicated that glycerol kinase accepts a range of substituents in place of one terminal hydroxyl group (which is not phosphorylated), and that the hydrogen atom at C-2 can be replaced by a methyl group. Replacement of the other (phosphorylated) hydroxyl group by other nucleophilic centers usually resulted in loss of activity. Comparisons of kinetic constants for enzymes from four microorganisms (Candidi mycoderma, Saccharomyces cerevisiae, Escherichia coli and Bacillus stearothermophilus) indicated little variation among them. All phosphorylated products had stereochemistry analogous to that of sn-glycerol-3-phosphate.
Chapter Two illustrates the use of glycerol kinase in synthesis, and provides experimental details for representative procedures on 10-500 mmol scale. Phosphorylation of racemic mixtures produced chiral organic phosphates with enantiomeric excess (%ee) > 95. The unphosphorylated enantiomer could be recovered from the reaction mixture in 30-40% yield and 80-95 %ee. Products produced include D-3-chloropropane-1,2-diol-1-phosphate, D-3-mercaptopropane-1,2-diol-1-phosphate, D-3-aminopropane-1,2-diol-3-phosphate, D-3-meth-oxypropane-1,2-diol-1-phosphate and D-butane-1,2,4-triol-1-phosphate. Preparations of sn-glycerol-3-phosphate (1 mol) and dihydroxyacetone phosphate (0.4 mol) are also described.
