Effect of Pressure on Deuterium Isotope Effects of Formate Dehydrogenase

Quirk, David J.; Northrop, Dexter B.

doi:10.1021/bi001991w

Cited by 23 publications

(12 citation statements)

References 34 publications

(53 reference statements)

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“…Although this model nicely fits the data of Isaacs, it only reproduced the data for MR when k TST (36)] as arising solely from within the transition state and ruled out a tunneling contribution to the reaction. Because we have strong evidence for a tunneling contribution to the reductive half-reaction of MR (13,42), we will attempt to use a full tunneling model to account for the observed pressure dependence of the KIE in MR.…”

Section: Discussionmentioning

confidence: 76%

“…Apart from temperature, the only other experimental parameter that has been exploited to study enzymatic H-tunneling reactions is that of hydrostatic pressure, an approach pioneered by Northrop in recent years (34)(35)(36)(37)(38). Semiclassical TST dictates that pressure effects are associated with differences in vibrational frequencies of isotopic and nonisotopic atoms (39,40).…”

mentioning

confidence: 99%

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

Hay

Sutcliffe

Scrutton

2007

Proc. Natl. Acad. Sci. U.S.A.

193

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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°C) and deuterium (12 to 31 s ؊1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°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.flavoprotein ͉ hydrogen tunneling ͉ morphinone reductase ͉ pressure dependence ͉ stopped flow E nzymes are efficient catalysts that can achieve rate enhancements of up to 10 21 over the uncatalyzed reaction rate (1). Our quest to understand the physical basis of this catalytic power, which is pivotal to our understanding of biological reactions and our exploitation of enzymes in chemical, biomedical, and biotechnological processes, is challenging and has involved sustained and intensive research efforts for Ͼ100 years (for reviews see, e.g., refs. 2-6). Recent experimental studies employing the temperature dependence of kinetic isotope effects (KIEs) have emphasized the role of quantum mechanical tunneling in enzymatic hydrogen-transfer (H-transfer) reactions (7-18). These data, which can be explained neither by conventional transition state theory (TST) nor by the Bell tunneling correction to TST (19), are in agreement with environmentally coupled models of hydrogen tunneling (H-tunneling) (12,20,21) and can be simulated computationally (22-30) within the framework of modern TST (31). In the Kuznetsov and Ulstrup environmentally coupled model of H-tunneling (20), as used quantitatively by Klinman and coworkers (32), temperatureindependent KIEs arise from Marcus-like (33) vibrations (collective thermally equilibrated motions) that lead to degenerate reactant and product states, whereas temperature-dependent KIEs arise from ''gating motions'' (motion along the reaction coordinate) that enhance the probability of tunneling at this configuration by ...

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Section: Discussionmentioning

confidence: 76%

mentioning

confidence: 99%

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

Hay

Sutcliffe

Scrutton

2007

Proc. Natl. Acad. Sci. U.S.A.

193

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“…Intrinsic KIEs were calculated from their observed values using a numerical solution of the modified Northrop equation: 34,35 …”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

et al. 2016

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The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system in studies of the role of protein dynamics in catalysis, but so far these studies were limited by the availability of structural information. Second, CbFDH and its mutants are of use in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in its commercial development. Here, we report the crystallization and structural determination of both holo-CbFDH and apo-CbFDH. The free energy barrier for the catalyzed reaction is computed, and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: The apo-enzyme crystal was obtained under co-crystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nM dissociation constant. It was found that the fraction of the apo-enzyme present in the solution is less than 1.7x10−7 (i.e. the solution is 99.9999% holo-enzyme). This is an extreme case where the crystal structure represents an insignificant fraction of enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

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“…In addition, the much larger positive activation volumes for sfDHFR at the lower (14.0˙0.1 mL mol 1 ) and higher Murakami et al (2010) c Ohmae et al (2013a) d Murakami et al (2011) e Ohmae et al (2012) (30.5˙0.2 mL mol 1 ) pressure regions suggest that the NADP C -releasing step and conformational changes such as partial unfolding, respectively, are the ratedetermining steps, because the two binding steps cannot be the rate-determining steps under saturated concentrations of DHF and NADPH (both 250 M). Conversely, when the activation volume is negative, the transition state has a smaller cavity and/or enhanced (i.e., higher) hydration than the ground state; this is the case when the chemical oxidation-reduction step is the rate-determining step (Northrop and Cho 2000;Quirk and Northrop 2001), because partial charges would be stabilized by the hydration or condensation of the hydrated water in the transitionstate structure.…”

Section: Pressure / Mpamentioning

confidence: 99%

Environmental Adaptation of Dihydrofolate Reductase from Deep-Sea Bacteria

Ohmae

Gekko

Kato

2015

Subcellular Biochemistry

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In order to elucidate the molecular adaptation mechanisms of enzymes to the high hydrostatic pressure of the deep sea, we cloned, purified, and characterized more than ten dihydrofolate reductases (DHFRs) from bacteria living in deep-sea and ambient atmospheric pressure environments. The nucleotide and amino acid sequences of these DHFRs indicate the deep-sea bacteria are adapted to their environments after the differentiation of their genus from ancestors inhabiting atmospheric pressure environments. In particular, the backbone structure of the deep-sea DHFR from Moritella profunda (mpDHFR) almost overlapped with the normal homolog from Escherichia coli (ecDHFR). Thus, those of other DHFRs would also overlap on the basis of their sequence similarities. However, the structural stability of both DHFRs was quite different: compared to ecDHFR, mpDHFR was more thermally stable but less stable against urea and pressure unfolding. The smaller volume changes due to unfolding suggest that the native structure of mpDHFR has a smaller cavity and/or enhanced hydration compared to ecDHFR. High hydrostatic pressure reduced the enzymatic activity of many DHFRs, but three deep-sea DHFRs and the D27E mutant of ecDHFR exhibited pressure-dependent activation. The inverted activation volumes from positive to negative values indicate the modification of their structural dynamics, conversion of the rate-determining step of the enzymatic reaction, and different contributions of the cavity and hydration to the transition-state structure. Since the cavity and hydration depend on amino acid side chains, DHFRs would adapt to the deep-sea environment by regulating the cavity and hydration by substituting their amino acid side chains without altering their backbone structure. The results of this study clearly indicate that the cavity and hydration play important roles in the adaptation of enzymes to the deep-sea environment.

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Effect of Pressure on Deuterium Isotope Effects of Formate Dehydrogenase

Cited by 23 publications

References 34 publications

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

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

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Environmental Adaptation of Dihydrofolate Reductase from Deep-Sea Bacteria

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