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Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

ABSTRACT: Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.

SUBMITTER: Martinez J

PROVIDER: S-EPMC4641920 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

Martinez Jacobo J Truffault Vincent V Hothorn Michael M

The Journal of biological chemistry 20150728 38

Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how ...[more]

PMID: 26221030

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Project description:Tauhe beta-phosphoglucomutase (beta-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of beta-glucose 1-phosphate (betaG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transfer from beta-glucose 1,6-(bis)phosphate (betaG1,6bisP) to betaG1P. Herein, we explore the mechanism by which hydrolysis of the beta-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate betaG1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 portion of the domain-domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by betaG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert betaG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray crystal structure of the T16P mutant at 2.7 A resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between betaG1,6bisP and betaG1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which betaG1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure.

Dataset Information

Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

Publications

Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

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