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Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy.

ABSTRACT: Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, ?(5)-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photoexcitation of a nitrile-containing photoacid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first step of the catalytic reaction. The nitrile of C183 is exposed to water when bound to the KSI active site, and we used time-resolved vibrational spectroscopy as a site-specific probe to study the solvation dynamics of water molecules in the vicinity of the nitrile. We observed that water molecules at the active site of KSI are highly rigid, during the light-activated catalytic cycle, compared to the solvation dynamics observed in bulk water. On the basis of this result, we hypothesize that rigid water dipoles at the active site might help in the maintenance of the preorganized electrostatic environment required for efficient catalysis. The results also demonstrate the utility of nitrile probes in measuring the dynamics of local (H-bonded) water molecules in contrast to the commonly used fluorescence methods which measure the average behavior of primary and subsequent spheres of solvation.

SUBMITTER: Jha SK

PROVIDER: S-EPMC3461217 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy.

Jha Santosh Kumar SK Ji Minbiao M Gaffney Kelly J KJ Boxer Steven G SG

The journal of physical chemistry. B 20120907 37

Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, Δ(5)-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photoexcitation of a nitrile-containing photoacid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first st ...[more]

PMID: 22931297

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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

Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy.

Publications

Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy.

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