TY - JOUR
T1 - Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase
AU - Jabalquinto, Ana María
AU - Laivenieks, Maris
AU - González-Nilo, Fernando D.
AU - Yévenes, Alejandro
AU - Encinas, María Victoria
AU - Zeikus, J. Gregory
AU - Cardemil, Emilio
N1 - Copyright:
Copyright 2004 Elsevier Science B.V., Amsterdam. All rights reserved.
PY - 2002/8
Y1 - 2002/8
N2 - 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.
AB - 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.
KW - Active site residues
KW - Anaerobiospirillum succiniciproducens
KW - Phosphoenolpyruvate carboxykinase
KW - Site-directed mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=52649090173&partnerID=8YFLogxK
U2 - 10.1023/A:1021178432158
DO - 10.1023/A:1021178432158
M3 - Article
C2 - 12492149
AN - SCOPUS:0036704099
VL - 21
SP - 393
EP - 400
JO - Protein Journal
JF - Protein Journal
SN - 1572-3887
IS - 6
ER -