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Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase.

ABSTRACT: A fundamental difference between enzymes and small chemical catalysts is the ability of enzymes to use binding interactions with nonreactive portions of substrates to accelerate chemical reactions. Remote binding interactions can localize substrates to the active site, position substrates relative to enzymatic functional groups and other substrates, trigger conformational changes, induce local destabilization, and modulate an active site environment by solvent exclusion. We investigated the role of remote substrate binding interactions in the reaction catalyzed by the enzyme ketosteroid isomerase (KSI), which catalyzes a double bond migration of steroid substrates through a dienolate intermediate that is stabilized in an oxyanion hole. Comparison of a single-ring and multiple-ring substrate allowed the catalytic contribution of binding interactions with the distal substrate rings to be determined. The value of k(cat)/K(M) for a single-ring substrate is reduced 27,000-fold relative to a multiple-ring steroid substrate, suggesting that remote binding interactions with the steroid substrate contribute substantially to the KSI reaction. Nevertheless, the reaction rates for KSI-bound single- and multiple-ring substrates (k(cat)) are within 2-fold. Further, oxyanion hole mutations have the same effect on reactions of the single- and multiple-ring substrates. These results suggest that remote binding interactions contribute >5 kcal/mol to catalysis by KSI but that local rather than remote interactions dictate the catalytic contributions from KSI's general base and oxyanion hole.

SUBMITTER: Schwans JP

PROVIDER: S-EPMC2732871 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase.

Schwans Jason P JP Kraut Daniel A DA Herschlag Daniel D

Proceedings of the National Academy of Sciences of the United States of America 20090812 34

A fundamental difference between enzymes and small chemical catalysts is the ability of enzymes to use binding interactions with nonreactive portions of substrates to accelerate chemical reactions. Remote binding interactions can localize substrates to the active site, position substrates relative to enzymatic functional groups and other substrates, trigger conformational changes, induce local destabilization, and modulate an active site environment by solvent exclusion. We investigated the role ...[more]

PMID: 19706511

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Project description:Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.

Dataset Information

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase.

Publications

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase.

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