PubMed Health⌕ Search

Biomedical subjects

V L Davidson

Publications and source records attributed to V L Davidson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Intermolecular electron transfer from substrate-reduced methylamine dehydrogenase to amicyanin is linked to proton transfer.

Within the methylamine dehydrogenase-amicyanin complex, intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) and copper. The ET reactions from two chemically distinct reduced forms of TTQ were studied. The quinol form of TTQ was generated by reduction by dithionite. An aminoquinol form of TTQ, in which an amino group displaces the carbonyl oxygen, was generated by reduction by the substrate methylamine. Thermodynamic analysis of the ET reactions suggested that the ET event was rate-limiting for the redox reaction between quinol TTQ and copper, but not for the ET reaction from aminoquinol TTQ to copper. Solvent kinetic isotope effect studies indicated that proton transfer was involved in the rate-limiting reaction step for the ET from the substrate-reduced enzyme, but not the dithionite-reduced enzyme. Solvent deuterium kinetic isotope effects of 1.5 and 12.2 were obtained, respectively, for the ET reactions from dithionite-reduced and substrate-reduced methylamine dehydrogenase. These results demonstrate that application of ET theory to the analysis of thermodynamic data for the intermolecular protein ET reactions can potentially be used to distinguish between true ET reactions and those which are gated or attenuated by adiabatic events. Kinetic models are presented to explain how the incorporation of the substrate-derived amino group into TTQ may alter the rate-limiting step for ET.

Bacterial Proteins↗
PubMed1995

Unusually large isotope effect for the reaction of aromatic amine dehydrogenase. A common feature of quinoproteins?

The reduction of the tryptophan tryptophylquinone [TTQ] prosthetic group of aromatic amine dehydrogenase by dopamine exhibits a deuterium kinetic isotope effect of 8.6-11.7. This effect is partially suppressed in the steady-state. These data support the proposed mechanism for the reductive half-reaction of AADH in which TTQ reduction is linked to proton abstraction from a covalent enzyme-substrate intermediate and in which release of the aldehyde product is partially rate-limiting for the overall reaction. The magnitude of this kinetic isotope effect appears to exceed the semiclassical limit for a proton abstraction. It is also nearly identical to values for the similar reactions catalyzed by methylamine dehydrogenase, which also possesses TTQ, and plasma amine oxidase, which possesses the topaquinone cofactor. These data suggest that these three quinoproteins share a very similar and possible unique mechanism for catalyzing the oxidative deamination of primary amines.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Mechanism of reaction of allylamine with the quinoprotein methylamine dehydrogenase.

Allylamine did not serve as an efficient substrate for methylamine dehydrogenase (EC 1.4.99.3) in a steady-state assay of activity and appeared to act as a competitive inhibitor of methylamine oxidation by methylamine dehydrogenase. Transient kinetic studies, however, revealed that allylamine rapidly reduced the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase. The rate of TTQ reduction by allylamine was 322 s-1, slightly faster than the rate of reduction by methylamine. These data were explained by a kinetic mechanism in which allylamine and methylamine are alternative substrates for methylamine dehydrogenase. The apparent competitive inhibition by allylamine is due to a very slow rate of release of the aldehyde product, 0.28 s-1, relative to a rate of 18.6 s-1 for the release of the aldehyde product of methylamine oxidation. A reaction mechanism is proposed for the oxidative deamination of allylamine by methylamine dehydrogenase. This mechanism is discussed in relation to the reaction mechanisms of topa-bearing quinoprotein amine oxidases, the flavoprotein monoamine oxidase and the mammalian semicarbazide-sensitive amine oxidase.

Allylamine↗
PubMed1995

Spectroscopic evidence for a common electron transfer pathway for two tryptophan tryptophylquinone enzymes.

Aromatic amine dehydrogenase (AADH) and methylamine dehydrogenase (MADH) are the only two enzymes known to use the cofactor tryptophan tryptophylquinone (TTQ). Each catalyzes oxidative deamination of a distinct class of primary amines. A detailed comparison of their circular dichroic spectra indicates that both proteins share a similar fold with their TTQ cofactors residing in similar environments and that this may be a useful diagnostic probe for TTQ enzymes. Alcaligenes faecalis cells induced to express AADH also express a large amount of the blue copper protein, azurin. Oxidized azurin is rapidly reduced by a catalytic amount of AADH in the presence of the substrate, tyramine. Three A. faecalis cytochromes-c and three other cytochromes-c were tested for electron transfer activity with AADH. Azurin markedly facilitated electron transfer from AADH to each cytochrome. This suggests that AADH and azurin may form an electron transfer complex with a c-type cytochrome, analogous to the crystallographically determined MADH-amicyanin-cytochrome c-551i complex (Chen, L., Durley, R. C. E., Matthews, F. S., and Davidson, V. L. (1994) Science 264, 86-90). The similarities of MADH and AADH plus the demonstration of azurin and multiple cytochromes as functional electron-transfer partners suggest that both TTQ-bearing enzymes share common mechanisms for oxidative deamination and subsequent electron transfer.

Alcaligenes↗
PubMed1995

Roles of dipolar effects and local charge in the ionic strength dependence of redox reactions between c-type cytochromes.

Redox reactions between different c-type cytochromes were monitored by stopped-flow spectroscopy. Second-order rate constants were determined at different ionic strengths for the reactions of Paracoccus denitrificans cytochrome c-551i with its physiologic redox partner cytochrome c-550, and with the nonphysiologic partner horse heart cytochrome c. The latter two cytochromes are structurally quite similar and exhibit identical redox potentials but bear net charges of -7 and +7, respectively. Despite these opposite overall charges, the ionic strength dependencies for the reaction of each with the acidic cytochrome c-551i were very similar. The observed decrease in reaction rate with increasing ionic strength that was observed with cytochromes c-550 and c-551i, the latter of which bears a net charge of -20, cannot be explained simply on the basis of monopole-monopole interactions. These data were analyzed by two different methods: one which treats proteins as both monopoles and dipoles and considers the net charge; and another which neglects dipolar effects and considers only the local charge of the reactive site of the protein rather than net charge. The applicability of each method to the analysis of these data and to protein electrostatic interactions in general is discussed.

Bacterial Proteins↗
PubMed1995

Mechanistic studies of aromatic amine dehydrogenase, a tryptophan tryptophylquinone enzyme.

Aromatic amine dehydrogenase (AADH) is the second enzyme known to possess the tryptophan tryptophylquinone (TTQ) prosthetic group. Its ability to catalyze the oxidative deamination of a wide range of aromatic and aliphatic amines has been investigated. Steady-state and transient kinetic studies of the reaction of AADH with a series of p-substituted phenylethylamines were performed to determine structure-reactivity correlations. The Km values correlated strongly with hydrophobic effects. The microscopic rate constant associated with TTQ reduction, k3, correlated with electronic substituent effects, particularly field/inductive effects, in a manner consistent with the formation of a carbanionic reaction intermediate in the reductive half-reaction. Transient kinetic studies were also performed with a series of p-substituted benzylamines, which were not substrates in the steady-state assay, but which did stoichiometrically reduce TTQ. The k3 for the reaction with benzylamines also correlated well with electronic effects. The rate constant associated with the release of the aldehyde product was also determined for the phenylethylamines and appears to be the most rate-limiting step in the overall oxidation-reduction reaction. This rate constant correlated with hydrophobic amines. This substrate specificity for aliphatic amines is opposite of that of methylamine dehydrogenase (MADH), the other known TTQ enzyme. On the basis of these studies, a reaction mechanism is proposed for AADH. These data are discussed in relation to the results of structure-reactivity correlation studies of the reactions catalyzed by MADH and two eukaryotic quinoproteins with different quinone prosthetic groups, plasma amine oxidase and lysyl oxidase.

Alcaligenes↗
PubMed1995

Amperometric detection of histamine at a quinoprotein dehydrogenase enzyme electrode.

Methylamine dehydrogenase, a tryptophan tryptophyl quinone (TTQ) containing quinoprotein, catalyzes the oxidation of a variety of primary aliphatic monomaines and diamines to their respective aldehydes and ammonia. This paper reports the construction and characterization of an enzyme electrode capable of detecting histamine and methylamine at +200 mV versus a saturated calomel reference electrode. The methylamine dehydrogenase isolated from Paracoccus denitrificans was used in conjunction with the insoluble mediator tetracyanoquinodimethane (TCNQ) to construct enzyme electrodes which will potentially provide simple rapid analysis of histamine without the need for the extensive sample pretreatments currently required in HPLC and GLC analysis. The linear response of this amperometric sensor, between 0 and 200 microM, correlates well with elevated histamine levels predominant in patients with chronic myelogenous leukaemia, whilst the observed limit of detection, 4.8 microM, compares favourably with the lower limits of detection reported for a potentiometric histamine sensitive enzyme electrode.

Biosensing Techniques↗
PubMed1994

Ionic strength dependence of the reaction between methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer.

The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1994

Thermal stability of methanol dehydrogenase is altered by the replacement of enzyme-bound Ca2+ with Sr2+.

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. At temperatures in the transition region for stability, the rate constants for inactivation of MEDH purified from these cells (Sr-MEDH) were 2-fold lower than those for MEDH. However, Arrhenius plots yielded an activation energy (Ea) of 699 kJ (167 kcal)/mol for MEDH compared with 640 kJ (153 kcal)/mol for Sr-MEDH. Further analysis by transition-state theory yielded values for the activation enthalpy (delta H*) and activation entropy (delta S*) of 696 kJ (166 kcal)/mol and 1.73 kJ (414 cal)/mol per K for MEDH and 637 kJ (152 kcal)/mol and 1.55 kJ (371 cal)/mol per K for Sr-MEDH. The higher rate of inactivation of MEDH than Sr-MEDH at higher temperatures is a consequence of a more favourable net gain in entropy. This positive entropy contribution increases at high temperatures, and reduces the more favourable stability obtained from the enthalpy contribution for the free energy (delta G*) of inactivation. The differences in these thermodynamic data are discussed in relation to the recently determined crystal structure of MEDH as well as 1H electron-nuclear double resonance studies of the influence of Sr2+ substitution on the structure of the pyrroloquinoline quinone-derived radical in MEDH.

Alcohol Oxidoreductases↗
PubMed1994

Kinetic and thermodynamic analysis of a physiologic intermolecular electron-transfer reaction between methylamine dehydrogenase and amicyanin.

The quinoprotein methylamine dehydrogenase (MADH) and a type I copper protein, amicyanin, form a physiologic complex in which electrons are transferred from tryptophan tryptophylquinone to copper. The reoxidation of MADH by amicyanin has been studied by stopped-flow spectroscopy. The rate constant for the electron-transfer (ET) reaction and the dissociation constant for the complex have been determined at different temperatures. Marcus theory was used to calculate the distance, reorganizational energy, and electronic coupling for the intermolecular ET reaction. The ET reaction exhibited a large apparent reorganizational energy of approximately 225 kJ mol-1 (2.3 eV) and a coupling of approximately 11.7 cm-1. From X-ray crystallographic studies of an actual complex of these proteins from Paracoccus denitrificans [Chen, L., et al. (1992) Biochemistry 31, 4959-4964], it was possible to infer putative pathways of ET. The ET distance predicted by Marcus theory from kinetic data correlated reasonably well with the structural information. Thus, it has been possible to correlate ET theories with data from solution studies and a known structure for a naturally occurring ET reaction between soluble proteins.

Bacterial Proteins↗
PubMed1994

Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol dehydrogenase.

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone (PQQ) prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. MEDH, which was purified from these cells (Sr-MEDH), exhibited an increased absorption coefficient for the PQQ chromophore, and displayed certain kinetic properties which were different from those of native MEDH. Native MEDH exhibits an endogenous activity which is not stimulated by substrate and which is inhibited by cyanide. Sr-MEDH exhibited lower endogenous activity which was stimulated by substrate, and was much less sensitive to inhibition by cyanide. The Vmax. for the methanol-dependent activity of Sr-MEDH was 3-fold greater than that of the native enzyme, and the Ks for methanol was altered. Cyanide also acts as an obligatory activator and competitive inhibitor of methanol-dependent activity in native MEDH from P. denitrificans [Harris and Davidson (1993) Biochemistry 32, 4362-4368]. Sr-MEDH exhibited a similar K1 for cyanide inhibition of methanol-dependent activity, but the KA for cyanide activation of this activity was 17-fold greater than that for the native enzyme. The activation energy of Sr-MEDH was 13.4 kJ (3.2 kcal)/mol lower than that of the native enzyme. These data confirm and significantly extend the conclusions from genetic [Richardson and Anthony (1992) Biochem. J. 287, 709-715] and crystallographic [White, Boyd, Mathews, Xia, Dai, Zhang and Davidson (1993) Biochemistry 32, 12955-12958] studies that suggest an apparently unique role for Ca2+ in MEDH compared with other Ca(2+)-dependent proteins and enzymes.

Alcohol Oxidoreductases↗
PubMed1994

Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i.

The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

Bacterial Proteins↗
PubMed1994

Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme.

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.

Alcaligenes↗
PubMed1993

Binding and electron transfer reactions between methanol dehydrogenase and its physiologic electron acceptor cytochrome c-551i: a kinetic and thermodynamic analysis.

The quinoprotein methanol dehydrogenase and cytochrome c-551i form a physiologic complex in which electrons are transferred from pyrroloquinoline quinone to heme. The reoxidation of methanol dehydrogenase by the cytochrome was studied by stopped-flow spectroscopy. The rate constant for the electron transfer reaction and the dissociation constant for complex formation were each determined at temperatures ranging from 20 to 50 degrees C. The electron transfer rates varied from 1.4 to 4.6 s-1. Analysis of the electron transfer reaction by Marcus theory yielded values of 1.9 eV for the reorganizational energy and 0.071 cm-1 for the electronic coupling and predicted a theoretical distance between redox centers of 15 A. Kinetically determined dissociation constants correlated well with a Kd of 375 microM which was determined in a direct ultrafiltration binding assay. Thermodynamic analysis of the dissociation constants indicated the importance of the hydrophobic effect in complex formation.

Alcohol Oxidoreductases↗
PubMed1993

The active site structure of the calcium-containing quinoprotein methanol dehydrogenase.

Pyrroloquinoline quinone (PQQ), widely found in nature, serves as the redox cofactor in bacterial methanol dehydrogenase (MEDH), a heterotetrameric enzyme that oxidizes methanol to formaldehyde. The refined structure of MEDH at 2.4-A resolution, based on recently obtained amino acid sequence data, reveals that the PQQ, located in a central channel of the disk-shaped protein, is sandwiched between a Trp side chain and a very unusual vicinal disulfide. A Ca2+ ion forms a bridge between PQQ and the protein molecule, very close to a putative substrate binding pocket. The vicinal disulfide may form during PQQ incorporation and possibly act to hold the latter in place.

Alcohol Oxidoreductases↗
PubMed1993

Binding constants for a physiologic electron-transfer protein complex between methylamine dehydrogenase and amicyanin. Effects of ionic strength and bound copper on binding.

Two soluble proteins, methylamine dehydrogenase and amicyanin, form a physiologically relevant complex in which intermolecular electron transfer occurs. To characterize and quantitate the binding of these two weakly-associating proteins, an ultrafiltration binding assay has been developed which involves brief centrifugation of mixtures of proteins in centrifuge concentrators followed by quantitation of proteins on each side of the filtration membrane by HPLC. Under low ionic strength conditions which are optimal for the redox reaction between these proteins, a Kd of 4.5 microM was measured for the methylamine dehydrogenase-amicyanin complex. The Kd increased by 8-fold in the presence of added salt. Apoamicyanin, which is known from crystallographic analysis to be structurally very similar to amicyanin, exhibited a much higher Kd and much less specific binding than did the holoprotein. Apoamicyanin also exhibited apparent self-association at low ionic strength which was not observed with amicyanin. These observations are correlated with the known crystal structures of these proteins, free and in complex, and with the available biochemical information on the interactions of these two proteins.

Apoproteins↗
PubMed1993

A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction.

The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.

Bacterial Proteins↗