Mechanism of the reductive half-reaction in cellobiose dehydrogenase

The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC ) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DH(cdh)) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DH(cdh) catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DH(cdh) is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DH(cdh) with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-A resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 A, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 A. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.     

The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: interaction with acyl-CoA analogues and an unusual thioesterase activity

A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acyl-CoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.     

Steady-state kinetic mechanism and reductive half-reaction of D-arginine dehydrogenase from Pseudomonas aeruginosa

D-arginine dehydrogenase from Pseudomonas aeruginosa catalyzes the oxidation of D-arginine to iminoarginine, which is hydrolyzed in solution to ketoarginine and ammonia. In the present study, we have genetically engineered an untagged form of the enzyme that was purified to high levels and characterized in its kinetic properties. The enzyme is a true dehydrogenase that does not react with molecular oxygen. Steady-state kinetic studies with D-arginine or D-histidine as substrate and PMS as the electron acceptor established a ping-pong bi-bi kinetic mechanism. With the fast substrate D-arginine a dead-end complex of the reduced enzyme and the substrate occurs at high concentrations of D-arginine yielding substrate inhibition, while the overall turnover is partially limited by the release of the iminoarginine product. With the slow substrate D-histidine the initial Michaelis complex undergoes an isomerization involving multiple conformations that are not all equally catalytically competent for the subsequent oxidation reaction, while the overall turnover is at least partially limited by flavin reduction. The kinetic data are interpreted in view of the high-resolution crystal structures of the iminoarginine--and iminohistidine--enzyme complexes.     

Interaction of acyl coenzyme A substrates and analogues with pig kidney medium-chain acyl-coA dehydrogenase

Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates

Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to betaAsp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product.     

Mitochondrial Acyl-CoA dehydrogenase activity of fish red muscle and pig liver

1. A method is described for the estimation of in vivo activity of mitochondrial Acyl-CoA dehydrogenase, without the addition of extra ETF (electron transferring flavoprotein). 2. Using Fumarase as a marker enzyme, the activity of Acyl-CoA dehydrogenase was calculated per gram wet weight. 3. The enzyme displays negative cooperativity in fish muscle, but positive cooperativity in pig liver. S0.5 values for the substrate octanoyl-CoA are between 3 and 5 microM.     

Electron-transferring flavoprotein from pig kidney: flavin analogue studies

Apo-electron-transferring flavoprotein from pig kidney (apo-ETF) has been prepared by an acid ammonium sulfate procedure and reconstituted with FAD analogues to probe the flavin binding site. The 8-position of the bound flavin is accessible to solvent as judged by the reaction of 8-Cl-FAD-ETF with sodium sulfide and thiophenol. A series of 8-alkylmercapto-FAD analogues containing increasingly bulky substituents bind tightly to apo-ETF and can be reduced to the dihydroflavin level by octanoyl-CoA in the presence of catalytic levels of the medium-chain acyl-CoA dehydrogenase. Bulky substituents severely slow the rate of these interflavin electron-transfer reactions. In the case of the 8-cyclohexylmercapto derivative, this decrease reflects a sizable increase in the Km for ETF (approximately 14-fold) with only a 20% decrease in Vmax. Reduction of all of these 8-substituted derivatives involves the accumulation of ETF anion radical intermediates. Dihydro-5-deaza-FAD dehydrogenase, unlike the corresponding 1-deazaflavin substitution, is unable to reduce native ETF despite a strongly favorable redox potential difference. These results, together with data from the native proteins, are consistent with obligatory 1-electron transfer between dehydrogenase and ETF possibly involving the exposed dimethylbenzene edge of ETF. Irradiation of apo-ETF reconstituted with the photoaffinity analogue 8-azidoflavin leads to approximately 10% covalent incorporation of the flavin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of apo-ETF labeled with tritiated 8-azido-FAD shows preferential labeling of the smaller subunit (88%, Mr 30,000 subunit; 12%, Mr 33,000 subunit).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver-Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.     

Redox potential and equilibria in the reductive half-reaction of Vibrio harveyi NADPH-FMN oxidoreductase

Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes.     

Purification and properties of pig kidney glutamate dehydrogenase.

Glutamate dehydrogenase from pig kidney has been purified to homogeneity by means of affinity chromatography on matrix bound Cibacron Blue F3G-A and gel chromatography on Sepharose 6B. The enzyme exhibits allosteric properties with the substrates alpha-ketoglutarate, ammonium, and NADH, respectively. GTP is a strong inhibitor which strengthened the cooperative interactions between the ammonium binding sites. ADP as an activator relieves the inhibition by GTP. Like glutamate dehydrogenase from bovine liver, glutamate dehydrogenase from pig kidney shows the ability of self-association, too. The sedimentation coefficient increases from 13.5 S at 0.07 mg protein/ml to 19.4 S at 1.32 mg protein/ml. In the sodium dodecylsulphate gel electrophoresis the enzyme migrates as a single band with a molecular-weight at 51000.     

Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH

Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.     

Kinetic studies on the broad-specificity beta-D-glucosidase from pig kidney.

A broad-specificity beta-D-glucosidase from pig kidney cortex was isolated and purified to homogeneity by a rapid purification procedure. The pI (5.14 +/- 0.05), Mr (59,000 +/- 2000) and specific activities with several p-nitrophenyl glycosides (galactopyranoside, glucopyranoside, arabinopyranoside, xylopyranoside) were comparable with those published previously for cytoplasmic beta-D-glucosidase from other sources and organs. Mixed-substrate experiments and inhibition studies with glucono-(1----5)-lactone revealed that a single active centre, containing one catalytic site and one saccharide-binding site, was responsible for the splitting of all four synthetic substrates. Inhibition experiments with substrate analogues demonstrated that (i) the major binding determinant of the glycosides was the aglycone moiety, (ii) an anionic side chain of the enzyme (probably a carboxy group) interacted with the glycosidic linkages and (iii) the properties of the aglycone significantly influenced the binding of the carbohydrate moiety. The inhibition constants of the p-nitrothiophenyl derivatives were in good agreement with the Km values of the corresponding substrates. Therefore the Michaelis constants could be regarded as true equilibrium constants (Ks). The 'three-point-attachment model' of the substrate splitting, proposed by Daniels [(1983) Ph.D. Dissertation, University of Pittsburgh] for the analogous liver enzyme, was applicable for beta-D-glucosidase from pig kidney too. The possible nature of the 'attachments' is discussed.     

The interaction of 2,3,4-trihydroxybenzylhydrazine with dopa decarboxylase from pig kidney

The spectral modifications induced by addition of 2,3,4,-trihydroxybenzylhydrazine (Ro 4-5127) to Dopa decarboxylase indicate the binding of this compound to the coenzyme binding site and allow the titration of the enzyme: 1.07 moles of Ro 4-5127 bind 1 mole of enzyme. Inhibition data indicate that Ro 4-5127 behaves as a pseudoirreversible inhibitor of Dopa decarboxylase and that 100% inhibition is not reached at a 1:1 inhibitor/ enzyme molar ratio, as expected from spectral data, but at a molar ratio of about 6. On this basis it would be possible to suggest, beside the coenzyme binding site, the existence on the enzyme molecule of other sites where the compound could bind without affecting its spectral feature. The interaction of Ro 4-5127 with Dopa decarboxylase shows that “in vivo” Ro 4-5127 behaves as a more powerful inhibitor of Dopa decarboxylase than its precursor trihydroxybenzylhydrazine seryl derivative (Ro 4-4602) indicating that the strong “in vivo” inhibition exerted by Ro 4-4602 might reflect the effective interaction of Dopa decarboxylase with Ro 4-5127.     

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅⁻, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH⁻, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.