Conformational flexibility of PEP mutase.

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TitleConformational flexibility of PEP mutase.
Publication TypeJournal Article
Year of Publication2004
AuthorsLiu, S, Lu, Z, Han, Y, Jia, Y, Howard, A, Dunaway-Mariano, D, Herzberg, O
JournalBiochemistry
Volume43
Issue15
Pagination4447-53
Date Published2004 Apr 20
ISSN0006-2960
KeywordsAlanine, Animals, Apoenzymes, Aspartic Acid, Binding Sites, Bivalvia, Crystallization, Crystallography, X-Ray, Magnesium, Models, Molecular, Mutagenesis, Site-Directed, Osmolar Concentration, Peptide Fragments, Phosphotransferases (Phosphomutases), Protein Conformation, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Substrate Specificity
Abstract

Previous work has indicated that PEP mutase catalyzes the rearrangement of phosphoenolpyruvate to phosphonopyruvate by a dissociative mechanism. The crystal structure of the mutase with Mg(II) and sulfopyruvate (a phosphonopyruvate analogue) bound showed that the substrate is anchored to the active site by the Mg(II), and shielded from solvent by a large loop (residues 115-133). Here, the crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are reported. In both unbound and Mg(II)-bound states, the active site is accessible to the solvent. The loop (residues 115-133), which in the enzyme-inhibitor complexes covers the active site cavity, is partially disordered or adopts a conformation that allows access to the cavity. In the apo state, the residues associated with Mg(II) binding are poised to accept the metal ion. When Mg(II) binds, the coordination is the same as that previously observed in the enzyme-Mg(II) sulfopyruvate complex, except that the coordination positions occupied by two ligand oxygen atoms are occupied by two water molecules. When the loop opens, three key active site residues are displaced from the active site, Lys120, Asn122, and Leu124. Lys120 mediates Mg(II) coordination. Asn122 and Leu124 surround the transferring phosphoryl group, and thus prevent substrate hydrolysis. Amino acid replacement of any one of these three loop residues results in a significant loss of catalytic activity. It is hypothesized that the loop serves to gate the mutase active site, interconverting between an open conformation that allows substrate binding and product release and a closed conformation that separates the reaction site from the solvent during catalysis.

DOI10.1021/bi036255h
Alternate JournalBiochemistry
PubMed ID15078090
Grant ListGM36260 / GM / NIGMS NIH HHS / United States