Conformational Flexibility of PEP Mutase

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• 作者：Sijiu Liu ; Zhibing Lu ; Ying Han ; Yong Jia ; Andrew Howard ; Debra Dunaway-Mariano ; and Osnat Herzberg
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：April 20, 2004
• 年：2004
• 卷：43
• 期：15
• 页码：4447 - 4453
• 全文大小：340K
• 年卷期：v.43,no.15(April 20, 2004)
• ISSN：1520-4995

文摘

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 theactive site by the Mg(II), and shielded from solvent by a large loop (residues 115-133). Here, the crystalstructures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are reported. Inboth unbound and Mg(II)-bound states, the active site is accessible to the solvent. The loop (residues115-133), which in the enzyme-inhibitor complexes covers the active site cavity, is partially disorderedor adopts a conformation that allows access to the cavity. In the apo state, the residues associated withMg(II) binding are poised to accept the metal ion. When Mg(II) binds, the coordination is the same asthat previously observed in the enzyme-Mg(II) sulfopyruvate complex, except that the coordinationpositions occupied by two ligand oxygen atoms are occupied by two water molecules. When the loopopens, 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 residuesresults in a significant loss of catalytic activity. It is hypothesized that the loop serves to gate the mutaseactive site, interconverting between an open conformation that allows substrate binding and product releaseand a closed conformation that separates the reaction site from the solvent during catalysis.