The effect of active site mutations in the oxaloacetate decarboxylase and pyruvate kinase-like activities of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase.
Anaerobiospirillum succiniciproducens His225Gln, Asp262Asn, Asp263Asn, and Thr249Asn phosphoenolpyruvate carboxykinases were analyzed for their oxaloacetate decarboxylase, and pyruvate kinase-like activities. The His225Gln and Asp263Asn enzymes showed increased Km values for Mn2+ and PEP compared with the native enzyme, suggesting a role of His225 and Asp263 in Mn2+ and PEP binding. No mayor alterations in Km values for oxaloacetate were detected for the varied enzymes. Alterations of His225, Asp262, Asp263, or Thr249, however, did not affect the Vmax of the secondary activities as much as they affected the Vmax for the main reaction. The results presented in this communication suggest different rate-limiting steps for the primary reaction and the secondary activities.
Jabalquinto
Ana María
AM
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile. ajabalqu@lauca.usach.cl
Laivenieks
Maris
M
Cabezas
Mauricio
M
Zeikus
J Gregory
JG
Cardemil
Emilio
E
D000244:Adenosine Diphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D002384:Catalysis; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics* / Q000378:metabolism*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism*; D020560:Proteobacteria / Q000201:enzymology* / Q000235:genetics; D011770:Pyruvate Kinase / Q000378:metabolism*
D008345:Manganese; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D019764:Phosphoenolpyruvate Carboxykinase (ATP)