Kinetic studies of formate dehydrogenase.
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ABSTRACT:
SUBMITTER: Peacock D
PROVIDER: S-EPMC1179669 | biostudies-other | 1970 Dec
REPOSITORIES: biostudies-other
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Project description:The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
| S-EPMC4917879 | biostudies-literature
Project description:Thermal motions of enzymes have been invoked to explain the temperature dependence of kinetic isotope effects (KIE) in enzyme-catalyzed hydride transfers. Formate dehydrogenase (FDH) from Candida boidinii exhibits a temperature independent KIE that becomes temperature dependent upon mutation of hydrophobic residues in the active site. Ternary complexes of FDH that mimic the transition state structure allow investigation of how these mutations influence active-site dynamics. A combination of X-ray crystallography, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamic simulations characterize the structure and dynamics of the active site. FDH exhibits oscillatory frequency fluctuations on the picosecond timescale, and the amplitude of these fluctuations correlates with the temperature dependence of the KIE. Both the kinetic and dynamic phenomena can be reproduced computationally. These results provide experimental evidence for a connection between the temperature dependence of KIEs and motions of the active site in an enzyme-catalyzed reaction consistent with activated tunneling models of the hydride transfer reaction.
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Project description:We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate C? hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.
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Project description:To define in detail the molecular mechanism of NAD+-dependent formate dehydrogenase, the pH dependences of various kinetic and spectroscopic parameters have been studied: Vmax, Km (NAD+), Km (formate), inhibition constants for structural analogues of substrate (NO3-) and product (CNS-, CNO-, N3-), CD and fluorescence properties. The value of Vmax, rate-limiting hydride transfer, is nearly constant throughout the entire pH range of enzyme stability (6.0-11.2) but decreases below 6. The K(m) values for both substrates remain constant within the pH range 6-10. At pH values below 6 (for the coenzyme) and above 10 (for both substrate and coenzyme) the Km values increase. In the acidic range this change is attributed to the ionization of two carboxy groups (pK approx. 5.5-6.0) located at the NAD+-binding site of the enzyme active centre. The pH transition in the basic region (pK 10.5 +/- 0.2) has a conformational origin and affects the enzyme's affinity for substrates and anion inhibitors. A similar transition has been observed for formate dehydrogenases from yeast Candida boidinii and Hansenula polymorpha. The results complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme.
| S-EPMC1218093 | biostudies-other
Project description:Isotopically labeled enzymes (denoted as "heavy" or "Born-Oppenheimer" enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born-Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein's macromolecular electrostatics at slower time scales; that is, both Born-Oppenheimer and non-Born-Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.
| S-EPMC5800309 | biostudies-literature
Project description:NAD(+)-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases.
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Project description:Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD(+) and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2.
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Project description:Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.
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Project description:Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7(T), expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions.
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Project description:Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.
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