Composition and Subunit Structure of Glycerol Kinase from Escherichia coli

Abstract

Abstract Glycerol kinase of Escherichia coli was purified and crystallized by a gentle modified procedure. A new assay utilizing [γ-32P]ATP was developed for following enzyme activity during purification. Electrophoresis in polyacrylamide gels and equilibrium ultracentrifugation of the crystalline enzyme indicated that this preparation was homogeneous. The amino acid composition of the enzyme was analyzed. Two independent tyrosine determinations by a colorimetric and a spectrophotometric method were in good agreement with the value from the amino acid analysis. The cysteine residues in the molecule are all present as the free sulfhydryls, with some residues exposed to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) only under denaturing conditions. The partial specific volume of the enzyme was found to be 0.732 ml per g from the amino acid composition or 0.724 ml per g by differential equilibrium sedimentation in buffers of H2O and D2O. With the use of these partial specific volumes, the molecular weight of the enzyme was determined by equilibrium ultracentrifugation to be 217,000 or 210,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, even without prior reduction, gave only one band of molecular weight 57,000, determined with respect to the migration of standard proteins. Equilibrium ultracentrifugation in the presence of 6 m guanidine hydrochloride indicated a homogeneous species of molecular weight 55,000. After reaction of the native enzyme with a bifunctional cross-linking reagent, dimethyl suberimidate, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yielded four bands, consistent with a tetrameric structure. Hydrazinolysis released only glutamic acid, in an amount equimolar to the subunit, suggesting that it is the only carboxyl-terminal amino acid. Mapping of the tryptic fragments of carboxymethylated enzyme yielded a number of ninhydrin-positive and of tyrosine- and tryptophan-containing peptides consistent with that expected from the amino acid analysis if the enzyme were composed of four identical subunits. It is concluded that glycerol kinase is an oligomeric protein composed of four identical subunits.