Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola.
The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.
Salvarrey
M S
MS
Cátedra de Química Biología, Facultad de medicina, Universidad de Buenos Aires, Paraguay, Argentina.
Cazzulo
J J
JJ
Cannata
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D002104:Cadmium / Q000494:pharmacology; D019256:Cadmium Chloride; D002245:Carbon Dioxide / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002413:Cations, Divalent / Q000494:pharmacology*; D002712:Chlorides / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D010071:Oxaloacetates / Q000378:metabolism*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000187:drug effects / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*
D002250:Carbon Radioisotopes; D002413:Cations, Divalent; D002712:Chlorides; D017895:Manganese Compounds; D010071:Oxaloacetates; D012265:Ribonucleotides; D002104:Cadmium; D015636:Magnesium Chloride; D002245:Carbon Dioxide; D000255:Adenosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D019256:Cadmium Chloride; C025340:manganese chloride