Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.

Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis.

The three enzymes glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were isolated in high yield from extracts of Zymomonas mobilis. The principal steps in the isolation procedures involved the use of selected dye-ligand adsorbent columns, with affinity elution of two of the three enzymes. Glucokinase and fructokinase are dimeric proteins (2 X 33000 Da and 2 X 28000 Da respectively) and glucose-6-phosphate dehydrogenase is a tetramer (4 X 52000 Da). Some similarities in the structural and kinetic parameters of the two kinases were noted, but they have absolute specificity for their substrates. Fructokinase is strongly inhibited by glucose; otherwise non-substrate sugars had little effect on any of the three enzymes.

Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 6-phosphogluconate dehydratase from Zymomonas mobilis.

Using differential dye-ligand chromatography and affinity elution with a substrate analog, 6-phosphogluconate dehydratase (EC 4.2.1.12) has been isolated from extracts of Zymomonas mobilis in a one-step procedure with 50% recovery. The specific activity of freshly isolated enzyme was 245 units mg-1. The enzyme contains iron, and it is rapidly inactivated in oxidizing conditions. It is inhibited by glycerophosphates, most strongly by the D-alpha-isomer which structurally corresponds to half of the substrate molecule.