Phosphoenolpyruvate carboxykinase from the moderate halophile Vibrio costicola. Purification, physicochemical properties and the effect of univalent-cation salts.
Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophilic bacterium Vibrio costicola. The enzyme is monomeric, with an Mr of 62,000, as determined by the Svedberg equation, by using values of s0(20,w) 4.4 x 10(-13) s, D20,w 6.13 x 10(-7) cm2.s-1 and v 0.719 cm3.g-1. Compared with other, non-halophilic, PEPCKs, the enzyme from V. costicola had a significantly lower total content of hydrophobic amino acids. The contents of glycine and serine were higher in the V. costicola enzyme (16.7 and 10.22% respectively) than in the non-halophilic PEPCKs (6.8-9.6% and 4.67-6.28% respectively). These results resemble those obtained by De Médicis & Rossignol [(1979) Experientia 35, 1546-1547] with the pyruvate kinase from V. costicola, and agree with the proposal by Lanyi [(1974) Bacteriol. Rev. 38, 272-290] of partial replacement of hydrophobic amino acids by glycine and serine to maintain the balance between hydrophobic and hydrophilic forces in halophilic enzymes. In agreement with this 'halophilic' characteristic, the PEPCK was somewhat stabilized by 1 M-KCl or -NaCl and by 20% (v/v) glycerol, and its oxaloacetate-decarboxylation and 14CO2-oxaloacetate-exchange reactions were activated by KCl and NaCl up to 1 M, whereas the fixation of CO2 on PEP had a maximum at 0.025-0.05 M salt. These facts suggest that the salts, at concentrations probably physiological for the bacterium, increase the formation of the complex of oxaloacetate and ATP with the enzyme, and the liberation of the products, PEP and ADP, thus favouring PEP synthesis.
Cátedra de Química Biológica, Facultad de Medicina, Universidad de Buenos Aires, Argentina.
M S
MS
Salvarrey
J J
JJ
Cannata
J J
JJ
Cazzulo
D000596:Amino Acids / Q000302:isolation & purification; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014733:Vibrio / Q000201:enzymology*
D000596:Amino Acids; D013439:Sulfhydryl Reagents; D012965:Sodium Chloride; D011189:Potassium Chloride; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005990:Glycerol