Structural and regulatory properties of pyruvate kinase from Pseudomonas citronellolis.

D.T Chuang,M.F Utter

doi:10.1016/s0021-9258(19)86910-7

Abstract

Pseudomonas citronellolis contains a single variety of pyruvate kinase regardless of the substrate on which the organism is grown. The enzyme has been purified to homogeneity and shown by cross-linking and gel electrophoretic studies to be a tetramer of M/sub r/ = 225,000 to 240,000. Unlike pyruvate kinases from yeast, liver, kidney, and red cells, the Pseudomonas enzyme is not affected by fructose 1,6-bisphosphate. However, it is activated by 2-keto-3-deoxy-6-phosphogluconate (KDPG), an intermediate of the Entner-Doudoroff pathway which is utilized for glucose catabolism by this organism. The Pseudomonas enzyme is also activated by ribose-5-P,5'-AMP, and fructose-6-P. Kinetic studies suggest that this group of metabolites may act at a single site with KDPG activation occurring at a separate site. All of the activators function by increasing the affinity of the enzyme for P-enolpyruvate. The latter substrate shows a strong positive cooperativity with the enzyme which is lowered or abolished by the presence of the activators. A strong synergistic effect exists between KDPG and ribose-5-P as exhibited by a mutual lowering of the concentrations required for activation as well as by a more than additive effect on the enzyme's affinity for P-enolpyruvate. Pyruvate kinase from P. citronellolis also differs from most varietiesmore » of the enzyme in that it is not activated by K/sup +/ orNH/sub 4//sup +/, requires high concentrations of Mg/sup 2 +/ and is relatively nonspecific with respect to its nucleoside diphosphate substrate.« less

Highlights

Pyruvate kinase from P. citronellolis differs from most varieties of the enzyme in that it is not activated by K’ or
It is interesting that the single species of Pseudomonas pyruvate kinase is activated by a key intermediate of each of these metabolic pathways, e.g. KDPG and ribose-5P, respectively
Ribose-5-P has a higher affinity for the enzyme than does KDPG, it is not clear that the former plays a more important regulatory role

Summary

PROCEDURES

5-P, Fru-Ps, 6-phosphogluconate, and all other sugar phosphates were obtained from Sigma Co. Frozen cells (approximately 75 g) of P. citronellolis grown on glucose were thawed in 320 ml of the extraction buffer containing, in millimolar concentrations: potassium phosphate (pH 7.2), 100; EDTA, 5; MgCb, 1; dithioerythritol, 2; and PMSF, 2 X 10m3. The precipitate was dissolved in a small volume of the elution buffer containing, in millimolar concentrations: Tris-Cl (pH 8.0), 25; sucrose, 300; PMSF, 2 x 10e3; and dithioerythritol, 2. Fractions containing the enzyme of specific activity of 2 units/mg or higher were pooled and precipitated with ammonium sulfate. 0.090 M KCl. Fractions which contained pyruvate kinase at specific activity of 10 unita/mg or higher were pooled and precipitated with ammonium sulfate. The precipitate was dissolved in 5 mM potassium phosphate buffer (pH 6.5) containing 0.3 M sucrose and 2 mM dithioerythritol.

RESULTS

DISCUSSION

Findings

D T Chuang and M F Utter