Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase.
Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.
Jabalquinto
Ana María
AM
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Laivenieks
Maris
M
González-Nilo
Fernando D
FD
Yévenes
Alejandro
A
Encinas
María Victoria
MV
Zeikus
J Gregory
JG
Cardemil
Emilio
E
D001665:Binding Sites; D002942:Circular Dichroism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020560:Proteobacteria / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D019764:Phosphoenolpyruvate Carboxykinase (ATP)