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Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects.

ABSTRACT: Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s(-1) at 25 degrees C) and deuterium (12 to 31 s(-1) at 25 degrees C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25 degrees C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of "barrier engineering" by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.

SUBMITTER: Hay S

PROVIDER: S-EPMC1766415 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects.

Hay Sam S Sutcliffe Michael J MJ Scrutton Nigel S NS

Proceedings of the National Academy of Sciences of the United States of America 20070103 2

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer r ...[more]

PMID: 17202258

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Project description:CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Project description:Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P?F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F?O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P?F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F?O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

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Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects.

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Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects.

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