Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.

[1]  P. Frey,et al.  The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer. , 1998, Biochemistry.

[2]  T. Nakano,et al.  Critical role of phenylalanine 34 of human dihydrofolate reductase in substrate and inhibitor binding and in catalysis. , 1994, Biochemistry.

[3]  B. Vainshtein,et al.  X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate. , 1996, European journal of biochemistry.

[4]  C. Chothia.  Structural invariants in protein folding , 1975, Nature.

[5]  Robert M. Smith,et al.  NIST Critically Selected Stability Constants of Metal Complexes Database , 2004 .

[6]  J. Zeikus,et al.  Evaluation by Site-Directed Mutagenesis of Active Site Amino Acid Residues of Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase , 2002, Journal of protein chemistry.

[7]  L. Prasad,et al.  The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3). , 2001, Journal of molecular biology.

[8]  B. Clantin,et al.  Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase , 2001, The EMBO journal.

[9]  Wu,et al.  Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases. , 1999, Structure.

[10]  S. Goldberg,et al.  Chemical equilibrium and reaction models , 1995 .

[11]  L. Delbaere,et al.  Snapshot of an enzyme reaction intermediate in the structure of the ATP–Mg2+–oxalate ternary complex of Escherichia coli PEP carboxykinase , 1996, Nature Structural Biology.

[12]  D. Parker,et al.  GEOCHEM‐PC—A Chemical Speciation Program for IBM and Compatible Personal Computers , 1995 .

[13]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[14]  T. Poulos,et al.  The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution. , 1994, The Journal of biological chemistry.

[15]  P. Frey,et al.  Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity , 2001, FEBS letters.

[16]  H. Michel,et al.  Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site. , 2002, Journal of molecular biology.

[17]  S. Trapani,et al.  Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution. , 2001, Journal of molecular biology.

[18]  L. Tong,et al.  Structural studies of the pigeon cytosolic NADP+‐dependent malic enzyme , 2002, Protein science : a publication of the Protein Society.

[19]  L. Delbaere,et al.  Mg2+–Mn2+ clusters in enzyme-catalyzed phosphoryl-transfer reactions , 1997, Nature Structural Biology.

[20]  J. Zeikus,et al.  Characterization of the Oxaloacetate Decarboxylase and Pyruvate Kinase-like Activities of Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinases , 1999, Journal of protein chemistry.

[21]  L. Delbaere,et al.  Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold. , 1996, Journal of molecular biology.

[22]  L. Delbaere,et al.  Structure and Mechanism of Phosphoenolpyruvate Carboxykinase* , 1997, The Journal of Biological Chemistry.

[23]  C. Vieille,et al.  Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene , 1997, Applied and environmental microbiology.

[24]  V. Schramm,et al.  Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate. , 1990, The Journal of biological chemistry.