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Identifies the structural functionalities of substrate or product that modulate the thermodynamic properties of medium-chain acyl-CoA dehydrogenase (MCAD); pH dependence of uncomplexed MCAD; Thioether-CoA-complexed MCAD; Acetyl-CoA-complexed MCAD.

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 reveal 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 Thrl 68Ala 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 into the catalytic mechanism can be obtained from simulation studies, and the results can be used to design novel mechanistic probes for the enzyme. [ABSTRACT FROM AUTHOR]

Natural substrate/product binding activates medium-chain acyl-CoA dehydrogenase (MCAD) to accept electrons from its substrate by inducing a positive flavin midpoint potential shift. The energy source for this activation has never been fully elucidated. If ground-state alterations of the ligand, such as polarization, are entirely responsible for enzyme activation, the ligand potential should shift equally to that of the flavin but in the opposite direction. Ligand polarization is likely responsible for only a small portion of this activation. Here, thiophenepropionoyl- and furylpropionoyl-CoA analogs were used to directly measure the redox modulations of several ligand couples upon binding to MCAD. These measurements identified the thermodynamic contribution of ligand polarization to enzyme activation. Because the ligand potential alterations are significantly smaller than modulations in the flavin potential due to binding, other phenomena such as pKa changes, desolvation, and charge alterations are likely responsible for the thermodynamic modulations required for MCAD''s activity. [Copyright &y& Elsevier]

Examines the biochemical behavior of the acyl-CoA dehydrogenase family toward the molecular oxygen. Suppression of reactivity of reduced enzyme toward molecular oxygen; Why a series of redox inactive acyl-CoA analogues was tested; Information on acyl-CoA dehydrogenase.

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial β-oxidation cycle tpr substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 my) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation. [ABSTRACT FROM AUTHOR]

Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial β-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. Two common variants of this enzyme encoding G185S and R147W substitutions have been identified at an increased frequency compared to the general population in patients with a wide variety of clinical problems, but functional studies of the purified mutant enzymes have shown only modestly changed kinetic properties. Moreover, both amino acid residues are located quite far from the catalytic pocket and the essential FAD cofactor. To clarify the potential relationship of these variants to clinical disease, we have further investigated their thermodynamic properties using spectroscopic and electrochemical techniques. Purified R147W hSCAD exhibited almost identical physical and redox properties to wild-type but only half of the specific activity and substrate activation shifts observed in wild-type enzyme. In contrast, the G185S mutant proved to have impairments of both its kinetic and electron transfer properties. Spectroelectro-chemical studies reveal that 0185S binding to the substrate/product couple produces an enzyme potential shift of only +88 mV, which is not enough to make the reaction thermodynamically favorable. For wild-type hSCAD, this barrier is overcome by a negative shift in the substrate/product couple midpoint potential, but in G185S this activation was not observed. When 0185S was substrate bound, the midpoint potential of the enzyme actually shifted more negative. These results provide valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrate that mutations, regardless of their position in the protein structure, can have a large impact on the redox properties of the enzyme. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase is a heterodimeric (α2β2) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (−163 ± 3 mV) different from that of R2Met produced by hydroxyurea (−115 ± 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming −223 ± 6 and −226 ± 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to −192 ± 2 mV for dGDP/dTTP and to −203 ± 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35–1.45 mM) and dATP (0.3–0.6 mM) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex. [ABSTRACT FROM AUTHOR]

The role of the oxyanion hole in the reaction catalyzed by pig medium-chain acyl-CoA dehydrogenase (pMCAD) has been investigated using enzyme reconstituted with 2'-deoxy-FAD. The k[sub cat] (18.8 ± 0.5 s[sup -1]) and K[sub m] (2.5 ± 0.4 µM) values for the oxidation of n-octanoyl-CoA (C[sub 8]-CoA) by WT pMCAD recombinantly expressed in Escherichia coli are similar to those of native pMCAD isolated from pig kidney. In agreement with previous studies [Engst et al. (1999) Biochemistry 38, 257-267], reconstitution of the WT enzyme with 2'-deoxy-FAD causes a large (400-fold) decrease in k[sub cat] but has little effect on K[sub m]. To investigate the molecular basis for the alterations in activity resulting from changes in hydrogen bonding between the substrate and the enzyme's oxyanion hole, the structure of the product analogue hexadienoyl-CoA (HD-CoA) bound to the 2'-deoxy-FAD-reconstituted enzyme has been probed by Raman spectroscopy. Importantly, while WT pMCAD causes a 27 cm[sup -1] decrease in the vibrational frequency of the HD enone band, from 1595 to 1568 cm[sup -1], the enone band is only shifted 10 cm[sup -1] upon binding HD-CoA to 2'-deoxy-FAD pMCAD. Thus, removal of the 2'-ribityl hydroxyl group results in a substantial reduction in the ability of the enzyme to polarize the ground state of the ES complex. On the basis of an analysis of a similar system, it is estimated that ground state destabilization is reduced by up to 17 kJ mol[sup -1], while the activation energy for the reaction is raised 15 kJ mol[sup -1]. In addition, removal of the 2'-ribityl hydroxyl reduces the redox potential shift that is induced by HD-CoA binding from 18 to 11 kJ mol[sup -1]. Consequently, while ligand polarization caused by hydrogen bonding in the oxyanion hole is intimately linked to substrate turnover, additional factors must be responsible for ligand-induced changes in redox potential. Finally, while replacement of the catalytic base E376 with Gln abolishes the ability of the enzyme to catalyze substrate oxidation and to catalyze the exchange of the C[sub 8]-CoA α-protons with solvent deuterium, the 2'-deoxy-FAD-reconstituted enzyme catalyzes α-proton exchange at a rate (k[sub exc]) of 0.085 s[sup -1], which is only 4-fold slower than k[sub exc] for WT pMCAD (0.35 s[sup -1]). Thus, either the oxyanion hole plays only a minor role in stabilizing the transition state for α-proton exchange, in contrast to its role in substrate oxidation, or the value of k[sub exc] for WT pMCAD reflects a process such as exchange of the E376 COOH proton with solvent. [ABSTRACT FROM AUTHOR]

Spectroelectrochemical and off-resonance Raman indicate that substrate/product binding to medium-chain acyl–coenzyme A (CoA) dehydrogenase (pMCAD) results in ligand polarization and positive flavin potential shifts, which activate the enzyme for electron transfer. Bacterial short-chain acyl-CoA dehydrogenase (bSCAD) typically exhibits smaller potential shifts upon substrate/product binding that have not been linked to ligand polarization. To further investigate the roles of ligand binding and polarization in activation, several novel aromatic carboxyloyl–CoAs were designed. These analogs allowed for the first direct comparison of pMCAD and bSCAD mechanisms. The results indicate that pMCAD activation can occur without perceptible analog polarization. bSCAD data provide the first spectral evidence of ligand polarization. The potential alterations exhibited by ligand-bound bSCAD are smaller than those of pMCAD, while their directionality and magnitude suggest differing enzyme–analog interactions. Such data provide the first indication of variations in the activation mechanism of these enzymes, which were thought to be comparable in both structure and function. [Copyright &y& Elsevier]

Studies the effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase. Mutations at the aromatic residues; Electrochemical properties of the mutants; Disruption of the interactions; Formation of product.