Computational mechanistic study of human liver glycerol 3‐phosphate dehydrogenase using ONIOM method (original) (raw)
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Modelling the active site of glyceraldehyde-3 phosphate dehydrogenase with the LSCF formalism
Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta), 1999
In the framework of a theoretical approach to the relationship between structure and reactivity of the catalytic centers of enzymes, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) has been chosen as a model enzyme. In GAPDH, the proximity of His 176 increases the reactivity of Cys 149 at neutral pH; however, its presence alone is not sucient to explain the reactivity of the catalytic Cys. In order to determine which other interactions play an important role, a study of the geometric and electronic structure of the catalytic site has been made using a hybrid quantum mechanics/ molecular mechanics local self-consistent ®eld method. This allows the computation of the electronic properties of amino acid residues in subsystems in¯uenced by other parts of the macromolecule. The quantum subsystem was centered on the Cys 149 residue of GAPDH. The structures of GAPDH taken from the crystallographic database did not include hydrogen atoms and these had to be added taking into account the fact that, in the active site, His 176 has three tautomeric forms: d-His protonated, -His protonated and His . The results presented here suggest that the most stable HisF F FCys system in GAP-DH is a strongly hydrogen-bonded Cys À 149 aHis 176 ion pair.
The side chains of R269 and N270 interact with the phosphodianion of dihydroxyacetone phosphate (DHAP) bound to glycerol 3-phosphate dehydrogenase (GPDH). The R269A, N270A, and R269A/N270A mutations of GPDH result in 9.1, 5.6, and 11.5 kcal/mol destabilization, respectively, of the transition state for GPDH-catalyzed reduction of DHAP by the reduced form of nicotinamide adenine dinucleotide. The N270A mutation results in a 7.7 kcal/mol decrease in the intrinsic phosphodianion binding energy, which is larger than the 5.6 kcal/mol effect of the mutation on the stability of the transition state for reduction of DHAP; a 2.2 kcal/mol stabilization of the transition state for unactivated hydride transfer to the truncated substrate glycolaldehyde (GA); and a change in the effect of phosphite dianion on GPDHcatalyzed reduction of GA, from strongly activating to inhibiting. The N270A mutation breaks the network of hydrogen bonding side chains, Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120, which connect the dianion activation and catalytic sites of GPDH. We propose that this disruption dramatically alters the performance of GPDH at these sites.
Crystal Structures of Human Glycerol 3-phosphate Dehydrogenase 1 (GPD1)
Journal of Molecular Biology, 2006
Homo sapiens L-a-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD C and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded b-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the 3-NH C 3 group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.
Comparative Computational Approach To Study Enzyme Reactions Using QM and QM-MM Methods
ACS Omega
Choline oxidase catalyzes oxidation of choline into glycine betaine through a two-step reaction pathway employing flavin as the cofactor. On the light of kinetic studies, it is proposed that a hydride ion is transferred from α-carbon of choline/hydrated-betaine aldehyde to the N5 position of flavin in the rate-determining step, which is preceded by deprotonation of hydroxyl group of choline/hydrated-betaine aldehyde to one of the possible basic side chains. Using the crystal structure of glycine betaine−choline oxidase complex, we formulated two computational systems to study the hydride-transfer mechanism including main active-site amino acid side chains, flavin cofactor, and choline as a model system. The first system used pure density functional theory calculations, whereas the second approach used a hybrid ONIOM approach consisting of density functional and molecular mechanics calculations. We were able to formulate in silico model active sites to study the hydride-transfer steps by utilizing noncovalent chemical interactions between choline/betaine aldehyde and active-site amino acid chains using an atomistic approach. We evaluated and compared the geometries and energetics of hydride-transfer process using two different systems. We highlighted chemical interactions and studied the effect of protonation state of an active-site histidine base on the energetics of transfer. Furthermore, we evaluated energetics of the second hydridetransfer process as well as hydration of betaine aldehyde.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1989
Perdeuterated spin label (DSL) analogs of NAD +, with the spin label attached at either the C8 or N 6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity of coenzyme binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and confonnational changes of the DSL-NAD +-enzyme complex during the catalytic reaction. Cg-DSL-NAD ÷ and N6-DSL-NAD + showed 80 and 45% of the activity of the native NAD +, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism o! this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD + in t!~ree conditions: (1) DSL-NAD + freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD + tightly bound to enzyme subunits remote (58 A) from other NAD + binding sites, and (3) DSL-NAD + bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 .~ or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD + in these three environents and measurements of the binding constants, KI-K 4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD + to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraidehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 ,~) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD + complex. When glyceraldehyde-3-pbosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the pbosphoglyceryl residue and DSL-NAD +. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD +. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD + from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.
Theoretical chemistry studies on the catalytic mechanism of liver alcohol dehydrogenase
Journal of Molecular Catalysis
The catalytic mechanism of liver alcohol dehydrogenase is reviewed from molecular and electronic standpoints. Detailed molecular and electronic descriptions of the factors intervening in catalysis have been obtained from Monte Carlo simulations of water structure and fluctuations in the substrate channel, active site molecular dynamics simulations of the 294-298 loop in the coenzyme binding domain that help close the substrate channel and bind the coenzyme, self-consistent reaction field calculations of proton relay structures that shuttle a proton from the active site towards the protein surface and vice versa, and ab initio MO analytical gradient studies of the hydride transfer step. A synthetic view of these studies is presented in this paper.
Journal of the American Chemical Society, 2001
The quantum dynamics of the hydride transfer reaction catalyzed by liver alcohol dehydrogenase (LADH) are studied with real-time dynamical simulations including the motion of the entire solvated enzyme. The electronic quantum effects are incorporated with an empirical valence bond potential, and the nuclear quantum effects of the transferring hydrogen are incorporated with a mixed quantum/classical molecular dynamics method in which the transferring hydrogen nucleus is represented by a three-dimensional vibrational wave function. The equilibrium transition state theory rate constants are determined from the adiabatic quantum free energy profiles, which include the free energy of the zero point motion for the transferring nucleus. The nonequilibrium dynamical effects are determined by calculating the transmission coefficients with a reactive flux scheme based on real-time molecular dynamics with quantum transitions (MDQT) surface hopping trajectories. The values of nearly unity for these transmission coefficients imply that nonequilibrium dynamical effects such as barrier recrossings are not dominant for this reaction. The calculated deuterium and tritium kinetic isotope effects for the overall rate agree with experimental results. These simulations elucidate the fundamental nature of the nuclear quantum effects and provide evidence of hydrogen tunneling in the direction along the donor-acceptor axis. An analysis of the geometrical parameters during the equilibrium and nonequilibrium simulations provides insight into the relation between specific enzyme motions and enzyme activity. The donor-acceptor distance, the catalytic zinc-substrate oxygen distance, and the coenzyme (NAD + / NADH) ring angles are found to strongly impact the activation free energy barrier, while the donor-acceptor distance and one of the coenzyme ring angles are found to be correlated to the degree of barrier recrossing. The distance between VAL-203 and the reactive center is found to significantly impact the activation free energy but not the degree of barrier recrossing. This result indicates that the experimentally observed effect of mutating VAL-203 on the enzyme activity is due to the alteration of the equilibrium free energy difference between the transition state and the reactant rather than nonequilibrium dynamical factors. The promoting motion of VAL-203 is characterized in terms of steric interactions involving THR-178 and the coenzyme.
ACS Catalysis, 2020
The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD + :acetoacetate and NAD + :3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold-and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.
The Journal of Physical Chemistry B, 2010
Glycerol binding and the radical-initiated hydrogen transfer by the coenzyme B 12-independent glycerol dehydratase from Clostridium butyricum were investigated by using quantum mechanical/molecular mechanical (QM/MM) calculations based on the high-resolution crystal structure (PDB code: 1r9d). Our QM/MM calculations of enzyme catalysis considered the electrostatic coupling between the quantum-mechanical and molecular-mechanical subsystems and two alternative mechanisms. In addition to performing QM/MM calculations in the enzyme, we evaluated energetics along the same reaction pathway in aqueous solution modeled by the polarized dielectric and in the virtual enzyme site that included full steric component from the enzyme residues described by molecular mechanics but lacked the electrostatic contribution of these residues. In this way, we established significant enzyme catalytic effect with respect to reference reactions in both an aqueous solution and a nonpolar cavity. Structurally, four hydrogen bonds formed between glycerol and H164, S282, E435, and D447 anchor glycerol for hydrogen abstraction by thiyl radical on C433. These hydrogenbond partners orient glycerol molecule to facilitate the formation of the transition state for hydrogen abstraction from carbon C1. This reaction then proceeds with the activation free energy of 6.3 kcal/mol and the reaction free energy of 6.1 kcal/mol. The polarization effects imposed by these hydrogen bonds represent a predominant contribution to a 7.5 kcal/mol enzyme catalytic effect. These results demonstrate the importance of electrostatic catalysis and hydrogen-bonding in enzyme-catalyzed radical reactions and advance our understanding of the catalytic mechanism of B 12-independent glycerol dehydratases.
Biochemistry, 2005
Potential of mean force calculations have been performed on the wild-type medium chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of the active site of the enzyme reveals that an arginine residue (Arg256), conserved in the substrate binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme active. This active conformation was used in subsequent computations of the enzymatic reactions. It is known that the catalytic α,β-dehydrogenation of fatty acyl-CoAs consists of two C-H bond dissociation processes: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in the wild-type MCAD demonstrate that the reaction proceeds by a stepwise mechanism with a transient species. The activation barriers of the two steps differ by only ∼2 kcal/ mol, indicating that both may contribute to the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barriers can be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along the reaction path reveals that Arg256 plays a key role in maintaining the reaction-center hydrogen-bonding network involving the thioester carbonyl group, which stabilizes transition states as well as the intervening transient species. Mutation of this arginine residue to glutamine increases the activation barrier of the hydride transfer reaction by ∼5 kcal/mol, and the present simulations predict a substantial loss of catalytic activity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioester moiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. In contrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relative orientation of the substrate and the cofactor in the active site; as a result, this mutation has very little effect on the overall reaction barrier, and this is consistent with the experimental data. This study demonstrates that significant insights of the catalytic mechanism can be obtained by these simulated enzyme catalysis studies whose results can pave the way of designing novel mechanistic probes for the enzyme. Mitochondrial β-oxidation has been extensively studied in the past 15 years because a number of mutations in several of the enzymes involved are responsible for diseases related to fatty acid metabolism. Fatty acid metabolism occurs within the mitochondrial matrix and proceeds by a sequence of steps that remove two carbon units in each cycle. The process involves at