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V L Davidson

Publications and source records attributed to V L Davidson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Electron transfer from the aminosemiquinone reaction intermediate of methylamine dehydrogenase to amicyanin.

The tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH) is covalently modified by substrate-derived nitrogen during its two-electron reduction by methylamine to form an aminoquinol (N-quinol). An N-semiquinone, which retains the substrate-derived N, is the intermediate during the two sequential one-electron oxidations of N-quinol MADH by its physiologic electron acceptor, amicyanin. Electron transfer (ET) from N-quinol MADH to amicyanin is gated by the deprotonation of the substrate-derived amino group on TTQ in the enzyme active site, whereas ET reactions from dithionite-reduced quinol and semiquinone forms of MADH are rate-limited by the ET event. The ET reaction from the N-semiquinone intermediate is shown not to be gated, but rate-limited by the ET step. Marcus analysis of the reaction reveals that the ET reaction from the N-semiquinone MADH to amicyanin exhibits the same reorganizational energy and electronic coupling as do the ET reactions of the dithionite-reduced O-quinol and O-semiquinone forms. The rates of the ET reactions of these three different redox forms of MADH exhibit a DeltaG degrees dependence which is predicted by Marcus theory. The ET reaction of the N-semiquinone is relatively insensitive to pH and salt, and does not exhibit a primary kinetic solvent isotope effect over the range of pH and cation concentrations studied. These properties are similar to those of the ET reaction of quinol MADH and different from those of the gated reaction of N-quinol MADH, whose rate varies considerably with pH and concentrations of specific monovalent cations. Thus, the covalent incorporation of substrate-derived N into TTQ is not alone sufficient to cause gating of ET. It affects the rate and DeltaG degrees for the ET reaction from the TTQ semiquinone by altering its redox potential, but it does not alter the reorganizational energy and electronic coupling associated with ET from TTQ to amicyanin.

Bacterial Proteins↗
PubMed1998

Redox properties of tryptophan tryptophylquinone enzymes. Correlation with structure and reactivity.

The pH dependence of the redox potentials for the oxidized/reduced couples of methylamine dehydrogenase (MADH) and aromatic amine dehydrogenase (AADH) were determined. For each enzyme, a change of -30 mV/pH unit was observed, indicating that the two-electron transfer is linked to the transfer of a single proton. This result differs from what was obtained from redox studies of a tryptophan tryptophylquinone (TTQ) model compound for which the two-electron couple is linked to the transfer of two protons. This result also distinguishes the redox properties of the enzyme-bound TTQ from those of the membrane-bound quinone components of respiratory and photosynthetic electron transfer chains that transfer two protons per two electrons. This difference is attributed to the accessibility of TTQ to solvent in the enzymes. One of the quinol hydroxyls is shielded from solvent and thus is not protonated. The unusual property of TTQ enzymes of stabilizing the anionic form of the reduced quinol is important for the reaction mechanism of MADH because it allows stabilization of physiologically important reaction intermediates. Examination of the extent to which disproportionation of the MADH and AADH semiquinones occurred as a function of pH revealed that the equilibrium concentration of semiquinone increased with pH. This indicates that the proton transfer is linked to the semiquinone/quinol couple. Therefore, the quinol is singly protonated, and the semiquinone is unprotonated and anionic. It was also shown that the oxidation-reduction midpoint potential for AADH is 20 mV less positive than that of MADH over the range of pH values that was studied and that the TTQ semiquinone of AADH was less stable than that of MADH. This may be explained by differences in the active site environments of the two enzymes, which modulate their respective redox properties.

Benzoquinones↗
PubMed1998

Methylamine dehydrogenase is a light-dependent oxidase.

Quinoproteins may function either as oxidases or dehydrogenases, depending on the nature of their quinone prosthetic group. The tryptophan tryptophylquinone (TTQ)-linked methylamine dehydrogenase (MADH) is relatively inert towards O2 in its reduced form. It is shown that on exposure to long range UV (ultaviolet) light, MADH is oxidized in the presence of O2 and exhibits substrate-dependent steady-state oxidase activity. The effects of light are completely reversible, and oxidase activity is lost when the light is turned off. The light-dependent oxidation of MADH proceeds via a semiquinone intermediate which accumulates to near stoichiometric levels. The absorption of the light appears to provide energy to overcome a thermodynamic barrier to the transfer of an electron from TTQ to O2. These studies provide a basis for understanding what factors dictate whether an oxidoreductase is a dehydrogenase or an oxidase.

Light↗
PubMed1998

Site-directed mutagenesis of Phe 97 to Glu in amicyanin alters the electronic coupling for interprotein electron transfer from quinol methylamine dehydrogenase.

Conversion by site-directed mutagenesis of Phe 97 of amicyanin to Glu significantly decreases the rate constant for the electron-transfer reaction from the quinol form of methylamine dehydrogenase to amicyanin. It is shown that the DeltaG degrees and reorganizational energy (lambda) associated with the electron-transfer reaction are unaffected by the mutation and that the decrease in the electron-transfer rate is attributable completely to a decrease in the electronic coupling matrix element (HAB). Phe 97 is not a part of the predicted pathway for electron-transfer from the tryptophan tryptophylquinone cofactor of MADH to the copper of amicyanin. The most likely explanation for these results is that the mutation of this residue at the protein-protein interface causes an increase in the interprotein distance within the protein complex. The change in distance necessary to cause the observed reduction of HAB is calculated assuming a range of beta values, and assuming either solely a direct distance dependence or a pathway dependence, for the long-range electron-transfer reaction. Thermodynamic analysis of the association constants for complex formation reveal that the reaction with the mutant amicyanin exhibits a large positive change in heat capacity whereas this is not observed in the reaction with the wild-type. This may be explained by the replacement of a hydrophobic residue with a polar residue at what is normally a hydrophobic protein-protein interface. The impact of these results on possible explanations for the relatively large reorganizational energy associated with this interprotein electron-transfer reaction is also discussed.

Bacterial Proteins↗
PubMed1998

Identification of reaction products and intermediates of aromatic-amine dehydrogenase by 15N and 13C NMR.

13C- and 15N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains covalently bound substrate-derived N. These data are consistent with the Ping Pong kinetic mechanism and aminotransferase-type chemical reaction mechanism which have been previously proposed for AADH. Comparison of the 15N-NMR spectra of the aminoquinol TTQ intermediates of AADH and methylamine dehydrogenase (MADH) revealed that the substrate-derived aminoquinol N of AADH and MADH exhibited distinct 15N chemical shifts which are separated by approx. 7 p.p.m. In each case, the signal for the substrate-derived aminoquinol N appears optimally with short pulse delay and exhibits a relaxation time and chemical shift which are consistent with 15N covalently bound to an aromatic ring (i.e. aminoquinol) which is attached to a rigid protein matrix. The aminoquinol of AADH is less stable against reoxidation than that of MADH. These data suggest that differences in the active-site mediated electrostatic environments of the aminoquinol N in the respective enzymes may influence both the observed 15N chemical shift and the relative reactivities of the TTQ aminoquinols towards oxygen. These data also demonstrate the utility of 13C- and 15N-NMR spectroscopy as a tool for monitoring the intermediates and products of enzyme-catalysed transformations.

Alcaligenes↗
PubMed1998

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans has been refined at 1.75 A resolution utilizing the DNA-based protein sequence. The final model incorporates 8034 atoms per molecule, including 552 molecules of solvent, and gives an R-factor of 0.163. The molecule is an H2L2 hetero-tetramer containing a non-crystallographic 2-fold axis of symmetry. The 373-residue H subunit is folded into seven repeats of a four-stranded antiparallel beta-sheet motif, arranged in a propeller-like pattern about a pseudo-7-fold rotational axis of symmetry. Each L subunit contains 131 residues folded in a tight structure composed of five beta-strands in two sheets and crosslinked by six disulfide bonds. In addition there is an intrasubunit covalent linkage between two tryptophan side-chains that form the unique redox center, tryptophan tryptophylquinone (TTQ). The active site contains the O-6 carbonyl of TTQ, the side-chains of Asp32L Asp76L, Tyr119L and Thr122L, and two solvent molecules. A potential "gate" (Phe55H) separates the closed active-site cavity from a channel containing a group of highly ordered water molecules to bulk solvent. Phe55H and Tyr119L, and a number of neighboring oxygen atoms, may also provide a binding site for monovalent cations that are known to affect the reactivity and spectral properties of TTQ as well as the oxidative half reaction. The overall reaction has been dissected into a number of discrete steps that may require participation by several individual amino acid residues in the active site acting as general acids and bases.

Amino Acid Sequence↗
PubMed1998

Kinetic and chemical mechanisms for the effects of univalent cations on the spectral properties of aromatic amine dehydrogenase.

Univalent cations and pH influence the UV-visible absorption spectrum of the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH). Little spectral perturbation was observed when pH was varied in the absence of univalent cations. The addition of alkali metal univalent cations (K+, Na+, Li+, Rb+ or Cs+) to oxidized AADH caused significant changes in its absorption spectrum. The apparent Kd for each cation, determined from titrations of the spectral perturbation, decreased with increasing pH. Transient kinetic studies involving rapid mixing of AADH with cations and pH jump revealed that the rate of the cation-induced spectral changes initially decreased with increasing cation concentration to a minimum value, then increased with increasing cation concentration. A kinetic model was developed to fit these data, determine the true pH-independent Kd values for K+ and Na+, and explain the pH-dependence of the apparent Kd. A chemical reaction mechanism, based on the kinetic data, is presented in which the metallic univalent cation facilitates the chemical modification of the TTQ prosthetic group to form an hydroxide adduct which gives rise to the spectral change. Addition of NH4(+)/NH3 to AADH caused changes in the absorption spectrum which were very different form those caused by addition of the metallic univalent cations. The kinetics of the reaction induced by addition of NH4+/NH3 were also different, being simple saturation kinetics. Another reaction mechanism is proposed for the NH4+/NH3-induced spectral change that involves nucleophilic addition of the unprotonated NH3 to TTQ. The general relevance of these data and models to the physiological reactions of TTQ-dependent enzymes and to the roles of univalent cations in modulating enzyme activity are discussed.

Alcaligenes↗
PubMed1997

Catalytic role of monovalent cations in the mechanism of proton transfer which gates an interprotein electron transfer reaction.

Within the methylamine dehydrogenase (MADH)-amicyanin protein complex, long range intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) of MADH and the type I copper of amicyanin. The reoxidations of two chemically distinct reduced forms of TTQ were studied, a quinol (O-quinol) generated by reduction by dithionite and the physiologically relevant aminoquinol (N-quinol) generated by reduction by methylamine. The latter contains a substrate-derived amino group which displaces the C6 carbonyl oxygen on TTQ. ET from N-quinol MADH to amicyanin is gated by the transfer of a solvent exchangeable proton [Bishop, G. R., & Davidson, V. L. (1995) Biochemistry 34, 12082-12086]. The factors which influence this proton transfer (PT) reaction have been examined. The rate of PT increases with increasing pH and with increasing salt concentration. The salt effect is due to specific monovalent cations and is not a general ionic strength effect. The rate enhancements by pH and cations do not reflect an elimination of the PT step that gates ET. Over the range of pH from 5.5 to 9.0 and with cation concentrations from 0 to 200 mM, the observed rate of the redox reaction is still that of PT. This is proven by kinetic solvent isotope effect studies which show that a primary isotope effect persists even at the highest values of pH and cation concentration. A model is presented to explain how specific cations contribute to catalysis and influence the rate of PT in this reaction. The pH dependence is attributed to an ionizable group that is involved in cation binding. The effect of the cation is stabilization of a negatively charged reaction intermediate that is formed during the deprotonation of the N-quinol, and from which rapid ET to the copper of amicyanin occurs. The relevance of these findings to other enzymes which exhibit reaction rates that are influenced by monovalent cations is also discussed.

Bacterial Proteins↗
PubMed1997

Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis.

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99. Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.

Amino Acid Sequence↗
PubMed1996

Intramolecular electron transfer in trimethylamine dehydrogenase: a thermodynamic analysis.

Within the enzyme trimethylamine dehydrogenase [TMADH], intramolecular electron transfer occurs between a fully reduced covalently bound 6-S-cysteinylflavin [FMN] cofactor, and an oxidized iron-sulfur [4Fe-4S]2+ center. When the enzyme is reduced by substrate trimethylamine, the kinetics of this intramolecular electron transfer [ET] reaction are biphasic, suggesting that ET occurs via two alternative processes [Falzon, L., & Davidson, V.L. (1996) Biochemistry 35, 2445-2452]. The formation of the FMN semiquinone was monitored by stopped-flow spectroscopy, and the two rate constants for the biphasic reaction were determined at temperatures ranging from 12 to 37 degrees C. Analysis of these rate constants by ET theory yielded values of 2.2 eV for the reorganizational energy [lambda] associated with each reaction and electronic coupling [H(AB)] of 5.9 and 47 cm-1 for the slower and faster ET reactions, respectively. The analysis also predicted average theoretical distance between the two redox centers of 12.3 A for the slower reaction and 8.1 A for the faster reaction. These predicted distances correlate well with the known crystal structure of TMADH and the most efficient pathways for ET that were predicted from the known structure using the Greenpath program. This analysis suggests that for each reaction the ET event is rate-limiting, but coupled to a highly unfavorable non-ET process, and that binding of a second molecule of substrate to reduced TMADH decreases the efficiency of the intramolecular ET.

Binding Sites↗
PubMed1996

Evidence for a tryptophan tryptophylquinone aminosemiquinone intermediate in the physiologic reaction between methylamine dehydrogenase and amicyanin.

The tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH) is covalently modified by nitrogen during its two-electron reduction by methylamine to form an aminoquinol (N-quinol). It is possible, in vitro, to generate unmodified O-quinol and O-semiquinone forms of MADH with dithionite, as well as an N-semiquinone form which contains a substrate-derived nitrogen. Rapid-scanning stopped-flow spectroscopy and global kinetic analysis are used to demonstrate that N-semiquinone is a true physiologic reaction intermediate which accumulates during the two sequential one-electron oxidations of N-quinol MADH by amicyanin. In contrast, no detectable O-semiquinone accumulates during the two sequential one-electron oxidations of the O-quinol form of MADH by amicyanin. This is because the reaction of N-semiquinone with amicyanin is much slower (70 s-1 at 25 degrees C) than the reaction of O-semiquinone ( > 1000 s-1). These rate constants obtained from global analysis of the overall reaction are the same as those obtained when each semiquinone form was made in vitro and then mixed with oxidized amicyanin. The presence of 200 mM NH4Cl during the reaction of O-quinol MADH with amicyanin does not cause any detectable accumulation of a semiquinone species. Thus, the accumulation of the intermediate in the reactions of the N-quinol is not due to the influence of noncovalently bound ammonia at the active site of the O-semiquinone. These data indicate that the intermediate which accumulates during the complete oxidation of substrate-reduced N-quinol MADH is not the O-semiquinone, but the more slowly reacting N-semiquinone, and that the N-semiquinone is a physiologically relevant reaction intermediate. These results also provide good evidence in favor of an aminotransferase mechanism, as opposed to an imine elimination mechanism, for the reaction of MADH with substrate methylamine.

Ammonium Chloride↗
PubMed1996

Electron transfer from copper to heme within the methylamine dehydrogenase--amicyanin--cytochrome c-551i complex.

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution [Chen, L., Durley, R., Mathews, F. S., & Davidson, V. L. (1994) Science 264, 86-90] which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The electron transfer reaction from copper to heme within the protein complex has been characterized by transient kinetic and thermodynamic analysis. The rate of this electron transfer reaction is 87 s-1 at 30 degrees C, and it varied with temperature. The reaction exhibited a reorganizational energy (lambda) of 1.1 eV and an electronic coupling (H(AB)) of 0.3 cm-1. The results of these analyses also predict an electron transfer distance, depending upon the value of beta which is used, of 13-24 A. The larger value approximates the direct copper to heme distance observed in the crystal structure of the complex. The most efficient pathways for electron transfer were predicted from the crystal structure using the Greenpath program, and these predictions were correlated with the results of the solution studies of the electron transfer reaction. It is concluded that electron transfer is, in fact, rate limiting for the observed electron transfer reaction in solution and that the two redox centers are strongly coupled, given the distance which separates them.

Bacterial Proteins↗
PubMed1996

Enzymatic and electron transfer activities in crystalline protein complexes.

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme.

Bacterial Proteins↗
PubMed1996

Kinetic model for the regulation by substrate of intramolecular electron transfer in trimethylamine dehydrogenase.

The reaction of trimethylamine dehydrogenase (TMADH) with trimethylamine has been studied by rapid-scanning stopped-flow spectroscopy and steady-state kinetics. The covalently bound 6-S-cysteinylflavin mononucleotide (FMN) cofactor is initially reduced by substrate and exhibits a limiting first order rate constant of 230 s(-1) at pH 7.5 and 30 degrees C. One electron is then transferred intramolecularly from the reduced FMNH2 to the oxidized [4Fe-4S]2+ center. This reaction is biphasic, and the extent of the reaction which corresponds to the faster and slower rates is dependent upon the concentration of trimethylamine. The limiting first order rate constants are 160 and 4 s(-1). At low substrate concentrations, the faster rate is dominant, and at high substrate concentrations, the slower rate is dominant. These results are used to develop a model for the reductive half-reaction of TMADH in which two molecules of substrate bind to TMADH. One binds at the active site of oxidized TMADH and is converted to products. A second molecule binds but is not converted to products and influences the rate of intramolecular electron transfer. Analysis of the transient kinetic data yielded apparent dissociation constants for trimethylamine of 36 and 148 mu M, respectively, for binding to the catalytic and noncatalytic sites. Steady-state kinetic studies indicated substrate inhibition which was best described by a model in which binding of a second molecule of trimethylamine causes a 10-fold reduction in k(cat) from 11 to 1.1 s(-1). This suggests that, at high substrate concentrations, the rate of the intramolecular electron transfer reaction has become sufficiently slow to be at least partially rate-limiting for the steady-state reaction. These kinetic data are interpreted in the context of the known crystal structure of TMADH. The mechanistic implications regarding long range electron transfer and possible physiologic significance of these findings are discussed.

Electrons↗
PubMed1995

Complex formation with methylamine dehydrogenase affects the pathway of electron transfer from amicyanin to cytochrome c-551i.

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution (Chen, L., Durley, R., Mathews, F. S., and Davidson, V. L. (1994) Science 264, 86-90), which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The reduction of cytochrome by amicyanin within the complex in solution has been demonstrated using rapid scanning stopped-flow spectroscopy. Electron transfer from free, uncomplexed, amicyanin to cytochrome c-551i occurs much more rapidly but only to a very small extent because the reaction is thermodynamically much less favorable when amicyanin is not associated with MADH (Gray, K. A., Davidson, V. L., and Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990). These kinetic data suggest that amicyanin binding to cytochrome c-551i occurs at different sites when amicyanin is free and when it is in complex with MADH. A model for the interactions of these proteins is presented.

Bacterial Proteins↗
PubMed1995

Reaction mechanism for the inactivation of the quinoprotein methylamine dehydrogenase by phenylhydrazine.

Phenylhydrazine has previously been shown to be an irreversible inactivator of the tryptophan tryptophylquinone (TTQ) enzyme methylamine dehydrogenase [Davidson, V.L. and Jones, L.H. (1992) Biochim. Biophys. Acta 1121, 104-110]. Structure-reactivity correlations have been performed to elucidate the mechanism by which this inactivation occurs. The reactions of a series of p-substituted phenylhydrazines with methylamine dehydrogenase were examined. Correlation with electronic substituent effects was observed. A Hammett plot of the second order inactivation rate constants versus sigma p exhibited a positive slope. Plots of these rate constants against substituent constants which reflected either resonance or field/inductive parameters for each p-substituent indicated that the rate was primarily influenced by resonance electronic effects. A Brønsted plot of the inactivation rate constant against pKa of each substituted phenylhydrazine yielded a beta-value (slope) of 0.7. Based upon these results, a reaction mechanism is proposed for the inactivation of methylamine dehydrogenase by phenylhydrazines, and a structure is proposed for the putative transition state for the rate-limiting step in the overall processes of binding and adduct formation by phenylhydrazine. The relevance of these results to the process of imine formation between substrate amines and TTQ during the normal catalytic process is also discussed.

Indolequinones↗
PubMed1995

Electron transfer reactions between aromatic amine dehydrogenase and azurin.

Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed that the apparent Km for azurin decreased with increasing ionic strength. These results are the opposite of what was observed for the reaction between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyanin, despite the fact that in both cases the pairs of redox proteins are each acidic proteins. It was further demonstrated that azurin does not function as an effective electron acceptor for MADH, and that amicyanin does not function as an effective electron acceptor for AADH. Thus, while the two TTQ enzymes each use type I copper proteins as physiologic electron acceptors, there is a strong specificity for which copper protein serves as a redox partner. The kinetic parameters for the electron transfer reactions from reduced AADH to oxidized azurin were determined by stopped-flow spectroscopy. Different results were obtained depending upon whether AADH was reduced chemically with dithionite or with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (kET) at 30 degrees C were 4 and 102 s-1, respectively. Kinetically determined values of Kd also differed by a factor of 2.4. These data suggest that the incorporation of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent kET. The interaction between AADH and azurin was also quantitated using an ultrafiltration binding assay. This yielded a Kd of 300 microM for the AADH--azurin complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗