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Modeling temperature dependent kinetic isotope effects for hydrogen transfer in a series of soybean lipoxygenase mutants: The effect of anharmonicity upon transfer distance.

ABSTRACT: Soybean lipoxygenase-1 (SLO) catalyzes the oxidation of linoleic acid. The rate-limiting step in this transformation is the net abstraction of the pro-S hydrogen atom from the center of the 1,5-pentadienyl moiety in linoleic acid. The large deuterium kinetic isotope effect (KIE) for this step appears in the first order rate constant ((D)k(cat) = 81 ± 5 at T = 25 °C). Furthermore, the KIE and the rate for protium abstraction are weakly temperature dependent (E(A,D) - E(A,H) = 0.9 ± 0.2 kcal/mol and E(A,H) = 2.1 ± 0.2 kcal/mol, respectively). Mutations at a hydrophobic site about 13 Å from the active site Fe(III), Ile(553), induce a marked temperature dependence that varies roughly in accordance with the degree to which the residue is changed in bulk from the wild type Ile. While the temperature dependence for these mutants varies from the wild type enzyme, the magnitude of the KIE at 25 °C is on the same order of magnitude. A hydrogen tunneling model [Kuznetsov, A.M., Ulstrup, J. Can. J. Chem. 77 (1999) 1085-1096] is utilized to model the KIE temperature profiles for the wild type SLO and each Ile(553) mutant. Hydrogenic wavefunctions are modeled using harmonic oscillators and Morse oscillators in order to explore the effects of anharmonicity upon computed kinetic observables used to characterize this hydrogen transfer.

SUBMITTER: Meyer MP

PROVIDER: S-EPMC2995544 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Modeling temperature dependent kinetic isotope effects for hydrogen transfer in a series of soybean lipoxygenase mutants: The effect of anharmonicity upon transfer distance.

Meyer Matthew P MP Klinman Judith P JP

Chemical physics 20051201 1-3

Soybean lipoxygenase-1 (SLO) catalyzes the oxidation of linoleic acid. The rate-limiting step in this transformation is the net abstraction of the pro-S hydrogen atom from the center of the 1,5-pentadienyl moiety in linoleic acid. The large deuterium kinetic isotope effect (KIE) for this step appears in the first order rate constant ((D)k(cat) = 81 ± 5 at T = 25 °C). Furthermore, the KIE and the rate for protium abstraction are weakly temperature dependent (E(A,D) - E(A,H) = 0.9 ± 0.2 kcal/mol a ...[more]

PMID: 21132078

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Project description:The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317-334) that correlate well with trends in the corresponding enthalpic barriers for kcat. A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD (Ea(ksolv) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation (Ea(kcat) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced "protein quakes" as the origin of enthalpic barriers in catalysis.

Dataset Information

Modeling temperature dependent kinetic isotope effects for hydrogen transfer in a series of soybean lipoxygenase mutants: The effect of anharmonicity upon transfer distance.

Publications

Modeling temperature dependent kinetic isotope effects for hydrogen transfer in a series of soybean lipoxygenase mutants: The effect of anharmonicity upon transfer distance.

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