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Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.

ABSTRACT: The tryptophan synthase ?2?2 bi-enzyme complex catalyzes the last two steps in the synthesis of l-tryptophan (l-Trp). The ?-subunit catalyzes cleavage of 3-indole-d-glycerol 3'-phosphate (IGP) to give indole and d-glyceraldehyde 3'-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25Å-long tunnel, from the ?-subunit to the ?-subunit where it reacts with l-Ser in a pyridoxal 5'-phosphate-dependent reaction to give l-Trp and a water molecule. The efficient utilization of IGP and l-Ser by tryptophan synthase to synthesize l-Trp utilizes a system of allosteric interactions that (1) function to switch the ?-site on and off at different stages of the ?-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the ?- and ?-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the ?-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.

SUBMITTER: Dunn MF

PROVIDER: S-EPMC3702454 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.

Dunn Michael F MF

Archives of biochemistry and biophysics 20120202 2

The tryptophan synthase α2β2 bi-enzyme complex catalyzes the last two steps in the synthesis of l-tryptophan (l-Trp). The α-subunit catalyzes cleavage of 3-indole-d-glycerol 3'-phosphate (IGP) to give indole and d-glyceraldehyde 3'-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25Å-long tunnel, from the α-subunit to the β-subunit where it reacts with l-Ser in a pyridoxal 5'-phosphate-dependent reaction to give l-Trp and a water molecule. The efficient utilization ...[more]

PMID: 22310642

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Project description:Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Toxoplasma gondii , contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain. The prevalence of T. gondii infections across the world, especially for those immunocompromised, underscores the need to understand TS-DHFR enzyme function and to find new avenues to exploit for the design of novel antiparasitic drugs. As a first step, we have solved the first three-dimensional structures of T. gondii TS-DHFR at 3.7 Å and of a loop truncated TS-DHFR, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 Å. Distinct structural features of the TS-DHFR homodimer include a junctional region containing a kinked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The roles of these unique structural features were probed by site-directed mutagenesis coupled with presteady state and steady state kinetics. Mutational analysis of the crossover helix region combined with kinetic characterization established the importance of this region not only in DHFR catalysis but also in modulating the distal TS activity, suggesting a role for TS-DHFR interdomain interactions. Additional kinetic studies revealed that substrate channeling occurs in which dihydrofolate is directly transferred from the TS to DHFR active site without entering bulk solution. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Taken together, these structural and kinetic studies reveal unique, functional regions on the T. gondii TS-DHFR enzyme that may be targeted for inhibition, thus paving the way for designing species specific inhibitors.

Dataset Information

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.

Publications

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex.

Similar Datasets

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