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Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase.

ABSTRACT: Indole-3-glycerol phosphate synthase catalyzes the terminal ring closure step in tryptophan biosynthesis. In this paper, we compare the results from molecular dynamics (MD) simulations of enzyme-bound substrate at 298, 333, 363, and 385 K and the enzyme-bound intermediate at 385 K, solvated in TIP3P water box with a CHARMM force field. Results from MD simulations agree with experimental studies supporting the observation that Lys-110 is the general acid. Based on its location in the active site during the MD simulations, Glu-210 warrants classification as the general base instead of the previously proposed Glu-159. We find that the relative population of the reactive enzyme-substrate Michaelis conformers [near attack conformers (NACs)] with temperature correlates well (correlation coefficient of 0.96) with the relative activity of this thermophilic enzyme. At higher temperature, the enzyme-substrate electrostatic interaction favors the binding of the substrate in NAC conformation, whereas, at lower temperature, the substrate is distorted and bound in a nonreactive conformation. This change is reflected in the approximately 1,100-fold increase in population of NACs at 385 K relative to 298 K. The easily determined population of NACs at given temperature tells much about the thermophilic property of the enzyme. Thus, the hyperthermophilic enzyme has evolved to have optimum activity at high temperatures, and, with lowering of the temperature, the electrostatic interaction at the active site is enhanced and the structure is deformed. This model can be regarded as a general explanation for the activity of hyperthermophilic enzymes.

SUBMITTER: Mazumder-Shivakumar D

PROVIDER: S-EPMC521968 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase.

Mazumder-Shivakumar Devleena D Bruice Thomas C TC

Proceedings of the National Academy of Sciences of the United States of America 20040927 40

Indole-3-glycerol phosphate synthase catalyzes the terminal ring closure step in tryptophan biosynthesis. In this paper, we compare the results from molecular dynamics (MD) simulations of enzyme-bound substrate at 298, 333, 363, and 385 K and the enzyme-bound intermediate at 385 K, solvated in TIP3P water box with a CHARMM force field. Results from MD simulations agree with experimental studies supporting the observation that Lys-110 is the general acid. Based on its location in the active site ...[more]

PMID: 15452341

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Project description: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.

Project description:The structures of partially folded states appearing during the folding of a (betaalpha)(8) TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (betaalpha)(4) region, modest protection in the neighboring (betaalpha)(1-3) and (betaalpha)(5)beta(6) segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (betaalpha)(2-5)beta(6) region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C(alpha) Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.

Dataset Information

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase.

Publications

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase.

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