title: Importance of aspartate residues in balancing the flexibility and fine-tuning the catalysis of human 3-phosphoglycerate kinase.
abstract: The exact role of the metal ion, usually Mg(2+), in the catalysis of human 3-phosphoglycerate kinase, a well-studied two-domain enzyme, has not been clarified. Here we have prepared single and double alanine mutants of the potential metal-binding residues, D374 and D218. While all mutations weaken the catalytic interactions with Mg(2+), they surprisingly strengthen binding of both MgADP and MgATP, and the effects are even more pronounced for ADP and ATP. Thermodynamic parameters of binding indicate an increase in the binding entropy as a reason for the strengthening. In agreement with the experimental results, computer-simulated annealing calculations for the complexes of these mutants have supported the mobility of the nucleotide phosphates and, as a consequence, formation of their new interaction(s) within the active site. A similar type of mobility is suggested to be a characteristic feature of the nucleotide site of the wild-type enzyme, too, both in its inactive open conformation and in the active closed conformation. This mobility of the nucleotide phosphates that is regulated by the aspartate side chains of D218 and D374 through the complexing Mg(2+) is suggested to be essential in enzyme function.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary. matkovics.varga.andrea@ttk.mta.hu
authors.forename: Andrea
authors.initials: A
authors.lastname: Varga
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Palmai
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Gugolya
authors.forename: Éva
authors.initials: É
authors.lastname: Gráczer
authors.forename: Ferenc
authors.initials: F
authors.lastname: Vonderviszt
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
authors.forename: Erika
authors.initials: E
authors.lastname: Balog
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D001224:Aspartic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium