Extremely Large Isotope Effects in the Soybean Lipoxygenase-Linoleic Acid Reaction

Glickman, Michael H.; Wiseman, Jeffrey S.; Klinman, Judith P.

doi:10.1021/ja00081a060

Cited by 157 publications

(197 citation statements)

References 1 publication

(1 reference statement)

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“…As can be seen, the running average converges to a value in the range 80-100, with an average value of 88, which is in good agreement with the experimentally observed KIE. 13 The running average ratio defined in eq 5 has the following properties. As stated earlier, it is constructed from transmission coefficients of hydrogen and deuterium, and hence, each term in the summation in the numerator on the right side corresponds to the cumulated ratio of probabilities of finding one particle over another on the product side for each point on the reaction coordinate.…”

Section: Resultsmentioning

confidence: 99%

Hydrogen Tunneling in an Enzyme Active Site: A Quantum Wavepacket Dynamical Perspective

2008

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We study the hydrogen tunneling problem in a model system that represents the active site of the biological enzyme, soybean lipoxygenase-1. Toward this, we utilize quantum wavepacket dynamics performed on potential surfaces obtained by using hybrid density functional theory under the influence of a dynamical active site. The kinetic isotope effect is computed by using the transmission amplitude of the wavepacket, and the experimental value is reproduced. By computing the hydrogen nuclear orbitals (eigenstates) along the reaction coordinate, we note that tunneling for both hydrogen and deuterium occurs through the existence of distorted, spherical s-type proton wave functions and p-type polarized proton wave functions for transfer along the donor-acceptor axis. In addition, there is also a significant population transfer through distorted p-type proton wave functions directed perpendicular to the donor-acceptor axis (via intervening π-type proton eigenstate interactions) which underlines the three-dimensional nature of the tunneling process. The quantum dynamical evolution indicates a significant contribution from tunneling processes both along the donor-acceptor axis and along directions perpendicular to the donor-acceptor axis. Furthermore, the tunneling process is facilitated by the occurrence of curve crossings and avoided crossings along the proton eigenstate adiabats.

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Section: Resultsmentioning

confidence: 99%

Hydrogen Tunneling in an Enzyme Active Site: A Quantum Wavepacket Dynamical Perspective

2008

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“…33). A number of enzymes have been shown to have KIEs substantially larger than the limit of ϳ7 set by transition-state theory, and this has also been taken as evidence for hydrogen tunneling (34,35). In view of the extremely large rate constant of the reaction (see above), it was of interest to look for a KIE on the reduction of NADP ϩ by NADH on dI 2 ⅐dIII 1 complexes.…”

Section: Resultsmentioning

confidence: 99%

Fast Hydride Transfer in Proton-translocating Transhydrogenase Revealed in a Rapid Mixing Continuous Flow Device

Pinheiro¹,

Venning²,

Jackson³

2001

Journal of Biological Chemistry

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Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI 2 ⅐dIII 1 complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP ؉ is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI 2 ⅐dIII 1 complexes catalyze reduction of NADP ؉ by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s ؊1 , which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD ؉ ) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.Transhydrogenase is found in the inner membranes of animal mitochondria and in the cytoplasmic membranes of many bacteria. It couples the redox reaction between NAD(H) and NADP(H) to inward translocation of protons across the membrane (Eq. 1).The enzyme provides NADPH for biosynthesis and glutathione reduction, and in mitochondria, it also helps to control flux through the tricarboxylic acid cycle (1, 2). The relative simplicity of transhydrogenase, the emergence of methods for determining its rate of reaction in real time (3-6), and recent high resolution structural information (7-12) make it a good model for understanding the general principles of operation of conformationally coupled ion translocators. The enzyme is composed of three components. dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude into the mitochondrial matrix (or the bacterial cytoplasm), and dII spans the membrane. The intact enzyme is effectively a dimer of two dI⅐dII⅐dIII "trimers" (13,14), though there are species variations in the way the polypeptide chains are joined. The findings that the transfer of hydride-ion equivalents between the bound nucleotides on transhydrogenase is direct (3, 5) and that there is no exchange of the transferred hydride with water protons (15) together establish that coupling to proton translocation does not occur at the redox step. We proposed an NADP(H) binding change model in which NADP ϩ (or NADPH) from the solvent can only bind to (or leave from) an "open" state of the dIII component of the protein and in which the redox reaction can only take place in an "occluded" state. Association and dissociation of protons during translocation, gated by the redox state of the NADP(H), drives the protein between the open and occluded states (16, 7). Recent observations on pro...

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“…However, detailed studies of C-H activation in enzyme reactions have forced us to move beyond this simplistic view. As observed initially in the reaction catalysed by soybean lipoxygenase (SLO; Glickman et al 1994;Hwang & Grissom 1994) and, subsequently, in a wide range of other enzyme-catalysed C-H abstraction reactions (e.g. Basran et al 1999Basran et al , 2001Kohen et al 1999;Abad et al 2000;Harris et al 2000;Francisco et al 2002;Sikorski et al 2004), the value of A 1 /A 2 often lies considerably above unity.…”

Section: Temperature Dependence Of Isotope Effects As a Measure Of Hymentioning

confidence: 88%

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

Klinman

2006

Phil. Trans. R. Soc. B

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Early studies of enzyme-catalysed hydride transfer reactions indicated kinetic anomalies that were initially interpreted in the context of a 'tunnelling correction'. An alternate model for tunnelling emerged following studies of the hydrogen atom transfer catalysed by the enzyme soybean lipoxygenase. This invokes full tunnelling of all isotopes of hydrogen, with reaction barriers reflecting the heavy atom, environmental reorganization terms. Using the latter approach, we offer an integration of the aggregate data implicating hydrogen tunnelling in enzymes (i.e. deviations from Swain-Schaad relationships and the semi-classical temperature dependence of the hydrogen isotope effect). The impact of site-specific mutations of enzymes plays a critical role in our understanding of the factors that control tunnelling in enzyme reactions.

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Extremely Large Isotope Effects in the Soybean Lipoxygenase-Linoleic Acid Reaction

Cited by 157 publications

References 1 publication

Hydrogen Tunneling in an Enzyme Active Site: A Quantum Wavepacket Dynamical Perspective

Hydrogen Tunneling in an Enzyme Active Site: A Quantum Wavepacket Dynamical Perspective

Fast Hydride Transfer in Proton-translocating Transhydrogenase Revealed in a Rapid Mixing Continuous Flow Device

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

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