Biomedical subjects

C Veeger

Publications and source records attributed to C Veeger.

At least 55 records · Page 3Linked to original sources

PubMed1991

Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild type and mutated enzymes.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active-site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro451----Ala [corrected] showed the greatest deviation from wild type. The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis-trans conversion. From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450----Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols. The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852-862]. Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗

PubMed1991

Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.

Acetates↗

PubMed1990

Reaction pathways for biodehalogenation of fluorinated anilines.

Pathways for biodehalogenation of fluorinated aniline derivatives were investigated. Microsomal NADPH-dependent dehalogenation of fluoroanilines was shown to proceed by three different reaction pathways. The first route appeared to result in monooxygenation at a fluorinated position and release of the fluorine atom as a fluoride anion. The primary additional reaction product formed is the reactive quinoneimine, not the 4-hydroxyaniline. In NADPH-containing microsomal systems with 4-fluoro-substituted anilines, formation of the 4-hydroxyaniline derivative is observed because NADPH chemically reduces this quinoneimine metabolite. A second pathway for dehalogenation proceeds by protein binding of a fluoro-containing (semi)quinoneimine metabolite, the formation of which may result from the mono-oxygenase reaction (pathway 1) and/or from (re)oxidation of a hydroxyaniline metabolite by superoxide anion radicals produced by the microsomal system. This latter reaction pathway becomes more important with increasing number of fluoro-substituents in the fluoroaniline derivative. The higher ratio of fluoride anion formed to 4-hydroxyaniline derivative detected in incubations with liver microsomes from dexamethasone-treated rats, as compared to incubations with liver microsomes from control rats, can in part be explained by the higher production of superoxide anion radicals observed in the dexamethasone systems. The third mechanism was shown to proceed by formation of a hydroxylated metabolite that loses fluoride anion upon exposure to oxygen. The reactive intermediate formed upon oxygen exposure might be the semiquinoneimine which loses its fluorine atom as a fluoride anion upon dimerization or polymerization and/or protein binding. The fluorohydroxyanilines, in which the hydroxyl group is ortho or para with respect to the fluoro substituent, appear especially to be highly unstable and lose fluoride anion in the presence of oxygen. Finally, it is concluded that all three pathways for dehalogenation of fluorinated aniline derivatives are bioactivation pathways. The reactivity of the (semi)quinoneimines formed in these reactions is dependent on their substitution pattern and increases with increasing number of fluoro-substituents. Therefore, bioactivation for a series of fluorinated aniline derivatives, can be expected to vary with the substitution pattern and to increase with increasing number of halogen substituents.

Aniline Compounds↗

PubMed1990

Description of enzyme kinetics in reversed micelles. 1. Theory.

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis-Menten kinetics, enzymes following a ping-pong bi-bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions. Whereas kinetic parameters derived from double-reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.

Colloids↗

PubMed1990

Enzyme kinetics in reversed micelles. 2. Behaviour of enoate reductase.

Enoate reductase (EC 1.3.1.31) can stereospecifically reduce a variety of alpha,beta-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609-625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 microM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5-5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.

Colloids↗

PubMed1990

Enzyme kinetics in reversed micelles. 3. Behaviour of 20 beta-hydroxysteroid dehydrogenase.

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.

20-Hydroxysteroid Dehydrogenases↗

PubMed1989

Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated.

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.

Bacteria, Anaerobic↗

PubMed1989

On the role of phospholipids in the reconstituted cytochrome P-450 system. A model study using dilauroyl and distearoyl glycerophosphocholine.

The difference in pentoxyresorufin O-dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P-450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P-450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P-450 for the NADPH-cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho-dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P-450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P-450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P-450 molecule, but also on the efficiency of electron transfer from NADPH-cytochrome reductase to cytochrome P-450.

Animals↗

PubMed1989

The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii. Expression in Escherichia coli and activation and isolation of the protein.

The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.

Acetyltransferases↗

PubMed1989

The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. A reconsideration.

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously. From the effect of guanidine hydrochloride on the light-scattering of intact E2 it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of the original mass before the N----D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.

Acetyltransferases↗

PubMed1989

hyd gamma, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase.

Downstream of the genes for the structural alpha and beta subunits of the periplasmic Desulfovibrio vulgaris (Hildenborough) hydrogenase a DNA fragment was detected with sequence homology to these genes. This fragment was cloned in Escherichia coli and the nucleotide sequence was determined. A gene was detected on the fragment with coding capacity for a 65.8 kDa polypeptide, hyd gamma. The central part of hyd gamma has an unusually high degree of homology with the alpha subunit and the C-terminal part has similarity with the beta subunit. These results strongly suggest that the three genes for hyd gamma and the alpha and beta subunits derive from one common ancestor gene. We succeeded in the identification of the translational product of this gene in E. coli, but were unable to determine the function of hyd gamma after expression in E. coli.

Amino Acid Sequence↗

PubMed1988

Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2-oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H-NMR spectroscopy.

600 MHz 1H-NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine-proline-rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N-terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low-field region of the 1H-NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H-NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain.

Acetyltransferases↗

PubMed1988

Time-resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl-bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A similar rotation is found in E2, indicating an independent rotational mobility of the whole domain in the multimeric E2 core (1.6 MDa). No independent rotation of a single lipoyl subdomain (10 kDa) is observed. The E1 component, in contrast to the E3 component, shows interaction with the lipoyl domain.

Acetyltransferases↗

PubMed1988

Detergentless microemulsions as media for enzymatic reactions. Cholesterol oxidation catalyzed by cholesterol oxidase.

Catalytic activity and stability of cholesterol oxidase dissolved in ternary systems composed of n-hexane, isopropanol, and water were studied. The dependence of catalytic activity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first-order inactivation rate constant of cholesterol oxidase dissolved in n-hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ.mol-1, respectively. Preparative conversion of cholesterol to cholestenone catalyzed by cholesterol oxidase in n-hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.

1-Propanol↗

PubMed1988

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis.

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.

Acetyltransferases↗

PubMed1988

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 1. Investigations by cyclic voltammetry.

The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n-aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one- and two-electron-reduced forms or only the two-electron-reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second-order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 10(6)-10(7) M-1 s-1 and increase in the direction propyl viologen sulfonate----methyl viologen----di(n-aminopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate-determining step in the catalytic mechanism.

Catalysis↗

PubMed1988

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 2. Investigations by chronoamperometry.

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility).

Catalysis↗

PubMed1988

The role of MgATP hydrolysis in nitrogenase catalysis.

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.

Azotobacter↗