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

Abstract

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.

Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein

“…A similar accumulation of abundant but inactive ADHII has been reported previously in recombinant strains of Z. mobilis (27). Both of these enzymes are easily inactivated during purification (36,42), and it is likely that the abundance of these proteins reflects the resistance of an inactive form to proteolysis and turnover. Five ethanologenic enzymes (GAP, PGK, ADHII, ENO, and PGM) remained abundant even in the stationary phase, indicating resistance to proteolysis.…”

Comparative Analysis of Pyruvate Kinases from the Hyperthermophilic Archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the Hyperthermophilic Bacterium Thermotoga maritima

“…K ϩ and NH 4 ϩ . K ϩ -independent PKs have been reported for E. coli, Corynebacterium glutamicum, and Z. mobilis and for the archaeon T. tenax (18,22,29,38). PK sequences contain highly conserved K ϩ sites (residues 48 -52 and 79 -88 of the Pyrobaculum PK), including a glutamate in K ϩ -stimulated PKs, e.g.…”

“…Few nonallosteric PKs have also been described, e.g. M1 isoenzyme of vertebrates and the dimeric PKs from Schizosaccharomyces pombe and Zymomonas mobilis (15,21,22). More than 140 primary sequences of eukarya and bacteria are known.…”

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

“…Z. mobilis PGK was purified using Procion Navy HE-R as an adsorbent. Following affinity elution with PGK substrates [23], the sample was subjected to Sephacryl 200 gel filtration. The sample was then dialysed and applied to a Q-Sepharose ion-exchange column.…”