Purification and properties of phosphoglucomutase from Fleischmann's yeast.
1. A procedure has been described for the purification of the major isozyme of yeast phosphoglucomutase of highest known specific activity. 2. The native enzyme has a molecular weight of about 65400 and was found to be homogeneous as judged by sucrose density gradient centrifugation, gel filtration, electrophoresis on acrylamide gel and ultracentrifugal analysis. In the presence of denaturing agents such as guanidine hydrochloride or sodium dodecyl sulfate, the enzyme dissociated into 32000-molecular-weight subunits. 3. As isolated, the enzyme has one mole of phosphate bound per mole of enzyme. Preparations incubated with 1.0 mM EDTA in 10 mM citrate buffer, pH 5.5 and dialysed against 10 mM metal-free citrate buffer, pH 5.5, contain no intrinsically bound Zn2+ and were enzymically inactive but fully active in the presence of 5 mM Mg2+ and 84% as active with 0.5 mM Zn2+. Simultaneous presence of both ions at these concentrations did not enhance activity. Enzyme was completely and irreversibly inactivated by preincubation with Be2+. Inactive enzyme had one mole of Be2+ bound per mole of enzyme. 4. Enzyme exhibited "ping-pong" kinetics rather than "random sequential". Km values for glucose 1-phosphate and for glucose 1,6-bisphosphate were calculated to be 2.34 times 10(-5) M and 2.24 times 10(-6) M, respectively. Rate of enzyme phosphate turnover was studied with rapid-mixing technique. The rates of 32P release from 32P-labeled enzyme and its appearance as glucose 6-[32P]phosphate were comparable and remained unaffected by addition of glucose 1,6-bisphosphate.
D000596:Amino Acids / Q000032:analysis; D001608:Beryllium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010733:Phosphoglucomutase / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D002413:Cations, Divalent; D007527:Isoenzymes; D010733:Phosphoglucomutase; D008274:Magnesium; D015032:Zinc; D001608:Beryllium