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Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis.

ABSTRACT: Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the ?1?1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the ?1?1 and ?2?2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the ?1?1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the ?1?1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the ?1?1 and ?2?2 loops, and highlight the multiple roles that the ?1?1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site ?1?1 and ?2?2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the ?1?1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

SUBMITTER: Zaccardi MJ

PROVIDER: S-EPMC3945838 | biostudies-other | 2014 Mar

REPOSITORIES: biostudies-other

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Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis.

Zaccardi Margot J MJ O'Rourke Kathleen F KF Yezdimer Eric M EM Loggia Laura J LJ Woldt Svenja S Boehr David D DD

Protein science : a publication of the Protein Society 20140204 3

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functi ...[more]

PMID: 24403092

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Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis.

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Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis.

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