A role for protein dynamics in enzymatic catalysis of Raltegravir Raltegravir hydrogen transfer has received substantial scientific support but the connections between protein structure and catalysis remain to be established. geometries (including donor-acceptor distances) in the V203A enzyme complexed with NAD+ and 2 3 4 5 6 alcohol or 2 2 2 (decided at 1.1 ?) are very similar to those for the wild-type enzyme except that this introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest in contrast to previous studies that this diminished tunneling and decreased rate of hydride transfer (16-fold relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis. The contributions of protein dynamics to enzyme catalysis have been studied with great interest recently. Kinetic isotope effects provide evidence for quantum mechanical hydrogen tunneling for various enzymatic reactions and the hydrogen transfer could be facilitated by protein motions that shorten the hydrogen donor-acceptor distance (DAD).1?3 Fast protein motions could be coincidental coupled correlated or in thermal equilibrium with the reaction coordinate.4?10 The motions can involve the whole protein as amino acid residues distant from the active site Raltegravir can take action through connected networks.11?13 Structural kinetic and computational studies of enzymes that are perturbed by site-directed amino acid substitutions can provide fundamental information about the functions of protein motions in catalysis. Horse liver alcohol dehydrogenase (ADH EC 220.127.116.11) is a good subject for these studies because structures can be determined at atomic resolution and the Raltegravir catalytic mechanism is well-described. X-ray crystallography of alcohol dehydrogenase has identified some of the dynamics involved in catalysis. The enzyme Raltegravir undergoes a global conformational change when coenzyme and substrate analogues bind.14?16 X-ray crystallography also provides evidence for puckering of the reduced ring in ternary complexes with aldehyde analogues17 and of the oxidized ring in complexes with fluoro alcohols.18 Deformation of the nicotinamide ring may be important for the formation of the tunneling-ready state.3 19 Horse liver ADH as studied with its mutated forms also exhibits kinetic isotope effects consistent with hydrogen tunneling.22?25 Schwartz and co-workers proposed that thermal motions namely “protein-promoting vibrations” (PPV) of specific amino acid residues facilitate the chemical reaction by modulating the distance between substrates significantly affecting catalysis by lowering the height and shortening the width of the reaction barrier.4 26 The calculations identified Ser-144 Gly-181 Val-203 Gly-204 Val-207 Glu-267 Ile-269 and Val-292 as residues in a conserved evolutionary sequence that contributes to PPV. The I269S substitution in the adenine binding site produced large increases in steady-state kinetic constants and made hydride transfer rate-limiting for ethanol oxidation but with only a modest decrease in the rate constant for transfer.16 29 The V292S substitution in the nicotinamide binding site also produced large increases in steady-state kinetic constants and made hydride transfer rate-limiting but with only a 4 decrease in the rate constant for benzyl alcohol oxidation.25 The subjects of this study are Val-203 which contacts the nicotinamide ring and Val-207 which is in a hydrophobic cluster near Val-203. Val-207 is usually highly conserved in dimeric ADHs but the human class 1A and 1B Mbp isoenzymes and the herb enzymes have an alanine residue.30 We expected that this V207A substitution should alter rate-promoting vibrations by creating a cavity and interrupting dynamic interactions. Large to small substitutions within the hydrophobic cores of enzymes can lead to cavities local shifts or collapse in structures or introduction of water molecules.31?33 A valine to alanine substitution can decrease protein stability by ～2 kcal/mol which should have a large effect on protein dynamics.34 35 Previous work showed that substitution of Val-203 (with Leu Raltegravir Ala or Gly) in ADH significantly decreases the catalytic efficiency for benzyl alcohol oxidation and diminishes the hydrogen tunneling as compared to that of the reference F93W.