Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′‐phosphate (PLP)‐dependent D ‐ornithine 4,5‐aminomutase (OAM)‐catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff‐base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial “strain” energy on the orientation of the cyclic intermediate to control its trajectory. In addition the “strain” energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP‐dependent reactions.     

Deep tunneling dominates the biologically important hydride transfer reaction from NADH to FMN in morphinone reductase

The temperature dependence of the primary kinetic isotope effect (KIE), combined temperature-pressure studies of the primary KIE, and studies of the alpha-secondary KIE previously led us to infer that hydride transfer from nicotinamide adenine dinucleotide to flavin mononucleotide in morphinone reductase proceeds via environmentally coupled hydride tunneling. We present here a computational analysis of this hydride transfer reaction using QM/MM molecular dynamics simulations and variational transition-state theory calculations. Our calculated primary and secondary KIEs are in good agreement with the corresponding experimental values. Although the experimentally observed KIE lies below the semiclassical limit, our calculations suggest that approximately 99% of the reaction proceeds via tunneling: this is the first "deep tunneling" reaction observed for hydride transfer. We also show that the dominant tunneling mechanism is controlled by the isotope at the primary rather than the secondary position: with protium in the primary position, large-curvature tunneling dominates, whereas with deuterium in this position, small-curvature tunneling dominates. Also, our study is consistent with tunneling being preceded by reorganization: in the reactant, the rings of the nicotinamide and isoalloxazine moieties are stacked roughly parallel to each other, and as the system moves toward a "tunneling-ready" configuration, the nicotinamide ring rotates to become almost perpendicular to the isoalloxazine ring.     

Laser photoexcitation of NAD(P)H induces reduction of P450 BM3 heme domain on the microsecond time scale

We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and the ferric BM3 heme domain (Kd increased to 1404 microM) but enhanced the rate of formation of the FeII-CO complex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)H photoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route to probing the P450 catalytic cycle and its transient intermediates.     

Conformational dynamics of the cytochrome P450 BM3/N-palmitoylglycine complex: the proposed "proximal-distal" transition probed by temperature-jump spectroscopy

The ferric spin state equilibrium of the heme iron was analyzed in wild-type cytochrome P450 BM3 and its F87G mutant by using temperature (T)-jump relaxation spectroscopy in combination with static equilibrium experiments. No relaxation process was measurable in the substrate-free enzyme indicating a relaxation process with a rate constant>10,000 s(-1). In contrast, a slow spin state transition process was observed in the N-palmitoylglycine (NPG)-bound enzyme species. This transition occurred with an observed rate constant (298 K) of approximately 800 s(-1) in the wild-type, and approximately 2500 s(-1) in the F87G mutant, suggesting a significant contribution of the phenylalanine side chain to a reaction step rate limiting the actual spin state transition. These findings are discussed in terms of an equilibrium between different binding modes of the substrate, including a position 7.5 A away from the heme iron ("distal") and the catalytically relevant "proximal" binding site, and are in accordance with results from X-ray crystallography, NMR studies, and molecular dynamics simulations.     

Tunneling and classical paths for proton transfer in an enzyme reaction dominated by tunneling: oxidation of tryptamine by aromatic amine dehydrogenase

Proton tunneling dominates the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase. For reaction with the fast substrate tryptamine, a H/D kinetic isotope effect (KIE) of 55 +/- 6 has been reported-one of the largest observed in an enzyme reaction. We present here a computational analysis of this proton-transfer reaction, applying combined quantum mechanics/molecular mechanics (QM/MM) methods (PM3-SRP//PM3/CHARMM22). In particular, we extend our previous computational study (Masgrau et al. Science 2006, 312, 237) by using improved energy corrections, high-level QM/MM methods, and an ensemble of paths to estimate the tunneling contributions. We have carried out QM/MM molecular dynamics simulations and variational transition state theory calculations with small-curvature tunneling corrections. The results provide detailed insight into the processes involved in the reaction. Transfer to the O2 oxygen of the catalytic base, Asp128beta, is found to be the favored reaction both thermodynamically and kinetically, even though O1 is closer in the reactant complex. Comparison of quantum and classical models of proton transfer allows estimation of the contribution of hydrogen tunneling in lowering the barrier to reaction in the enzyme. A reduction of the activation free energy due to tunneling of 3.1 kcal mol-1 is found, which represents a rate enhancement due to tunneling by 2 orders of magnitude. The calculated KIE of 30 is significantly elevated over the semiclassical limit, in agreement with the experimental observations; a semiclassical value of 6 is obtained when tunneling is omitted. A polarization of the C-H bond to be broken is observed due to the close proximity of the catalytic aspartate and the (formally) positively charged imine nitrogen. A comparison is also made with the related quinoprotein methylamine dehydrogenase (MADH)-the much lower KIE of 11 that we obtain for the MADH/methylamine system is found to arise from a more endothermic potential energy surface for the MADH reaction.     

Proton tunneling in aromatic amine dehydrogenase is driven by a short-range sub-picosecond promoting vibration: consistency of simulation and theory with experiment

Hydrogen transfer, an essential component of most biological reactions, is a quantum problem. However, the proposed role of compressive motion in promoting enzymatic H-transfer is contentious. Using molecular dynamics simulations and density functional theory (DFT) calculations, we show that, during proton tunneling in the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase (AADH), a sub-picosecond promoting vibration is inherent to the iminoquinone intermediate. We show by numerical modeling that this short-range vibration, with a frequency of approximately 165 cm-1, is consistent with "gating" motion in the hydrogen tunneling model of Kuznetsov and Ulstrup (Kuznetsov, A. M.; Ulstrup, J. Can. J. Chem. 1999, 77, 1085) in an enzymatic reaction with an observed protium/deuterium kinetic isotope effect that is not measurably temperature-dependent.     

Protein motions are coupled to the reaction chemistry in coenzyme b(12) -dependent ethanolamine ammonia lyase

The role of protein dynamics in promoting catalysis is hotly debated. Infrared data from both ultrafast flash photolysis and stopped-flow studies show that not only does there appear to be vibrational coupling between the cofactor and protein in B(12) -dependent ethanolamine ammonia lyase, but also that there are significant protein motions coupled to the reaction that follows substrate binding.     

Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.