PubMed Health⌕ Search

Biomedical subjects

V Massey

Publications and source records attributed to V Massey.

At least 19 recordsLinked to original sources
PubMed1995

Chemical modification of the N-10 ribityl side chain of flavins. Effects on properties of flavoprotein disulfide oxidoreductases.

Three flavin derivatives modified at the 2'-position of the flavin N-10 ribityl side chain were synthesized: arabinoflavin, 2'-F-2'-deoxyarabinoflavin, and 2'-deoxyriboflavin. These were converted to the FAD level with FAD synthetase. Apoproteins of lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, a family of flavoprotein oxidoreductases, were reconstituted with these flavins. Significant reduction of the catalytic activities was observed with the modified enzymes. During anaerobic reduction of the modified enzymes with substrate or dithiothreitol, decreased thermodynamic stability of the two-electron reduced enzyme forms (EH2) and the accumulation of the four-electron reduced forms (EH4) noted. This effect was more pronounced in case of arabino-FAD-reconstituted enzymes than with the other two. It was found that NAD+ binding influences the interaction between the flavin and the reduced disulfide in the 2'-F-arabino-FAD-lipoamide dehydrogenase, presumably by altering the relative oxidation-reduction potentials. 19F NMR data were obtained for different forms of the 2'-F-arabino-FAD-lipoamide dehydrogenase, which suggest marked conformational changes from one form to the other. The 19F NMR data for the oxidized forms of all three 2'-F-arabino-FAD proteins suggest that the fluorine experiences very similar chemical environments at the active sites.

Animals↗
PubMed1995

Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component.

The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide. Both enzymes' Km values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately. Vmax values for hydrogen peroxide or cumene hydroperoxide reduction are > 10,000 min(-1) at 25 degrees C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C40SC337S/C340S. Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S). Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.

Amino Acid Sequence↗
PubMed1995

A site-directed mutagenesis study at Lys-113 of NAD(P)H:quinone-acceptor oxidoreductase: an involvement of Lys-113 in the binding of the flavin adenine dinucleotide prosthetic group.

NAD(P)H: quinone-acceptor oxidoreductase (EC 1.6.99.2), also referred to as DT-diaphorase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. Using an Escherichia coli expression system developed previously, we prepared three mutants of the rat liver quinone reductase. These mutants are Lys-113-His (K113H), Lys-113-Asp (K113D), and Lys-113-Ala (K113A). While the mutant K113H was readily purified using the same procedure as for the purification of the wild-type quinone reductase and found to have an activity similar to that of the wild-type enzyme, K113D and K113A were purified only in very small quantities, mainly in the form of apoprotein, and had very low activities. The results suggest that a positively charged amino acid at this position is important for the binding of the flavin adenine dinucleotide (FAD) prosthetic group. Flavin spectral studies of 6-mercapto-FAD-reconstituted mutants revealed that mutation at Lys-113 affects the protein environment around position-6 of the isoalloxazine ring.

Amino Acid Sequence↗
PubMed1995

2-Aminobenzoyl-CoA monooxygenase/reductase. Evidence for two distinct loci catalyzing substrate monooxygenation and hydrogenation.

2-Aminobenzoyl-CoA monooxygenase/reductase catalyzes both monooxygenation and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 substrate analogs has been investigated. Only 2-aminobenzoyl-CoA (anthraniloyl-CoA) in its normal and deuterated (5-2H) form is a full substrate, and only traces of 2-hydroxybenzoyl-CoA (salicyloyl-CoA) are probably monooxygenated but not hydrogenated. The purified enzyme is a homodimer and has been resolved preparatively into three major species by anion-exchange chromatography on Mono Q. All three species have the same specific activity when reconstituted to full content of FAD, they differ, however, substantially in their mode of binding FAD. The oxidized or fully reduced enzyme forms bind tightly 0.5 mol/mol of the substrate 2-aminobenzoyl-CoA (Kd = 1-2 microM). The enzyme can be depleted of approximately 50% of its FAD, which corresponds to essentially complete removal from one of the two binding sites, reflecting a large difference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads to loss of the hydrogenation capacity of the enzyme, while monooxygenation catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-ethylmaleimide. Exchange of reducing equivalents between the two FAD cofactors at the respective binding sites is very slow and irrelevant compared to the rates of catalysis. It is concluded that the enzyme, which has been proposed to consist of two identical polypeptide chains [Altenschmidt, U., Bokranz, M. & Fuchs, G. (1992) Eur. J. Biochem. 207, 715-722], contains two active centers which differ substantially in their catalytic activity. One center belongs to the class of monooxygenases, the other one to the (de)hydrogenases. This must result from substantially different interaction of the same flavin cofactors with protein functional groups and is, to our knowledge, unprecedented in flavoprotein enzymology.

Catalysis↗
PubMed1995

Old Yellow enzyme: aromatization of cyclic enones and the mechanism of a novel dismutation reaction.

The origin of charge transfer bands that develop on reaction of Old Yellow Enzyme with alpha,beta-unsaturated cyclic ketones such as 3-oxodecalin-4-ene (ODE, numbered according to the convention for steroids), 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC), and 2-cyclohexenone is shown to be due to the aromatization of ODE and ODEC to 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and of 2-cyclohexenone to phenol. The aromatization of ODEC to HTN is stereospecific and involves the trans dehydrogenation of the 1 beta, 2 alpha hydrogens. The aromatization occurs under aerobic as well as anaerobic conditions. With the exception of ODEC under aerobic conditions, the aromatization of these substrates is accompanied by a dismutation reaction in which the olefinic bond of a second molecule of each substrate is reduced to give the saturated cyclic ketone. Molecular oxygen may serve as the electron acceptor with ODEC and some other substrates under aerobic reaction conditions. The dismutation reaction involves an overall sequence of hydride transfer from one substrate molecule to the beta-carbon of a second substrate molecule along with a solvent proton uptake by the alpha-carbon. 19-Nortestosterone is aromatized to beta-estradiol; however, other 3-oxo-delta 4-steroids such as progesterone, testosterone, and androstene-3,17-dione bind tightly to the enzyme but are not aromatized. The NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds is limited to aldehydes and ketones. alpha,beta-Unsaturated acids, esters, amides, and nitriles are not reduced. The reduction of the olefinic bond of ODE or cinnamaldehyde by NADPH occurs by an overall sequence of hydride transfer from the reduced pyridine nucleotide to the beta-carbon of the alpha,beta-unsaturated carbonyl compound and a solvent proton uptake by the alpha-carbon. The 4-pro-R hydride of NADPH is transferred in the reduction reaction. Structure-function relationships in the NADPH-dependent reduction of alpha,beta-unsaturated aldehydes or ketones indicate that increasing alkyl substitution at the beta-carbon results in marked decrease in the rate of reduction of the olefinic bond, consistent with a steric hindrance to hydride transfer at the beta-carbon.

Acrolein↗
PubMed1995

Active site studies of DT-diaphorase employing artificial flavins.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.

Binding Sites↗
PubMed1995

A new old yellow enzyme of Saccharomyces cerevisiae.

In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V. (1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

Amino Acid Sequence↗
PubMed1995

DT-diaphorase. Redox potential, steady-state, and rapid reaction studies.

NAD(P)H:quinone oxidoreductase (DT-diaphorse) appears to be a 2-electron transfer flavoprotein, which catalyzes the conversion of quinones into hydroquinones. Upon photoreduction in the presence of dimethylformamide, the enzyme forms a red semiquinone. In the absence of dimethylformamide, only 10% of the radical form is thermodynamically stabilized. This indicates a redox potential of the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The 2-electron redox potential was determined to be -159 +/- 3 mV at 25 degrees C, pH 7.0. In the presence of benzoquinone or 3-aminopyridine adenine dinucleotide phosphate, as NADPH analogue, there is no change in the redox properties of the enzyme flavin. A significant decrease is observed in the presence of the competitive inhibitor dicumarol (Em = -234 +/- 2 mV at pH 7.0). The reaction mechanism of the flavoprotein has been investigated by steady-state and stopped-flow kinetic methods using NADPH, NADH, deamino-NADPH, and 3-acetylpyridine adenine dinucleotide reduced form (APADH) as electron donors and K3Fe(CN)6, 4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ), and 2,5-diaziridinyl-3,6-bis(carboethoxy-amino)-1,4-benzoquinone (AZQ) as electron acceptors in 50 mM phosphate buffer, pH 7.0, 25 degrees C. No evidence could be obtained to indicate that semiquinoid intermediates play a part in the catalytic mechanism of DT-diaphorase with quinones as acceptors. The rates of the reduction by NADPH, NADH, deamino-NADPH, and APADH (1.3 x 10(9), 8.8 x 10(8), 8.3 x 10(8) and 9.8 x 10(8) m-1 min-1, respectively) as well as the rates of the reoxidation by PQQ and AZQ (9 x 10(4) and 2.8 x 10(6) M-1 min-1, respectively) are directly proportional to substrate concentration, and there is no evidence of the formation of enzyme-substrate complexes. If such complexes do indeed exist, the affinity of the enzyme for substrate must be extremely low. Using K3Fe(CN)6 as electron acceptor, the rate of oxidation of fully reduced enzyme is 4.6 x 10(7) M-1 min-1 and it is accurately proportional to ferricyanide concentration. This rate represents that of flavin semiquinone formation, with the subsequent oxidation of the semiquinone being much faster, since no spectral evidence for semiquinone formation could be obtained. Studies were also conducted attempting to use apo-DT-diaphorase reconstituted with PQQ as coenzyme. The lack of activity toward AZQ, K3Fe(CN)6, and menadione suggests that DT-diaphorase can use PQQ only as electron acceptor and not as redox cofactor.

Flavin-Adenine Dinucleotide↗
PubMed1994

Studies on the kinetic mechanism of pig kidney D-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228.

Expression conditions in Escherichia coli of wild-type, Y224F, and Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 totally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate. Reoxidation of the reduced flavin results from reaction of oxygen with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.

Animals↗
PubMed1994

The mobile flavin of 4-OH benzoate hydroxylase.

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Benzoate 4-Monooxygenase↗
PubMed1994

Studies of the reductive half-reaction of milk xanthine dehydrogenase.

The reductive half-reaction of milk xanthine dehydrogenase (XDH) with NADH and with xanthine has been studied at pH 7.5, 25 degree C. NADH reduces XDH to the two-electron reduced form at a rate of 18 s-1, independent of NADH concentration over the range studied. Further reduction by NADH to the four-electron state is inhibited by excess NADH. Subsequent binding of NADH to the four-electron reduced form of the enzyme causes the redistribution of one electron from the flavin to the molybdenum center. The four-electron reduced species reached through reduction by NADH is the same as the species obtained upon reaction of NAD with fully reduced XDH. In contrast, xanthine rapidly reduces XDH to the four-electron level; further reduction is comparatively slow and is inhibited by excess xanthine. Studies using substoichiometric xanthine show that the reaction of XDH with 1 equivalent of xanthine involves rapid substrate binding and rapid reduction of the molybdenum center of the enzyme. Before the release of urate from the molybdenum active site, an electron is transferred at 15 s-1 from the reduced molybdenum center to one of the iron-sulfur centers of XDH. Urate is then released at a rate of 13 s-1, followed by a rapid electron redistribution within the protein. The reductive half-reaction of XDH with xanthine is rate-limiting in xanthine/NAD turnover, which appears to occur between the two- and four-electron reduced enzyme species. The reduction of XDH by substoichiometric amounts of the fluorescent substrate xanthopterin was also studied. This reaction, monitored by changes in both absorbance and fluorescence, was found to involve the formation of two molybdenum complexes (an Eox.S complex and an Ered.P complex) followed by the release of the product, leucopterin.

Animals↗
PubMed1992

p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme.

p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein. The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2. A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation. Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein. A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity. The coupling protein does not seem to have any redox center in it. Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD.

Anaerobiosis↗
PubMed1992

Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.

Animals↗
PubMed1992

Direct expression of active spinach glycolate oxidase in Escherichia coli.

Spinach glycolate oxidase (GAO) was expressed in Escherichia coli using the T7 RNA polymerase promotor. The enzyme accounts for approx. 1% of the soluble protein fraction and is expressed as a soluble and active enzyme. Comparison with GAO expressed in Saccharomyces cerevisiae (Macheroux, P., Massey, V., Thiele, D.J. and Volokita, M. (1991) Biochemistry 30, 4612-4619) showed that the GAO expressed in E. coli has identical physico-chemical features to the wild-type enzyme, but is expressed at a level approx. 15-fold higher than in the yeast system.

Alcohol Oxidoreductases↗
PubMed1992

Light product of photoreactive 6-azido-FAD bound to deflavo-milk xanthine oxidase.

Xanthine oxidase from milk was reconstituted with the photoreactive flavin, 6-azido-FAD. While irradiation of the reconstituted enzyme under anaerobic conditions yielded 6-amino-FAD as a light product, aerobic irradiation resulted in formation of an unknown product, which gave the enzyme almost the same activity as that of the native enzyme. The light product could be extracted from the enzyme without breakdown and was found to be highly fluorescent. Upon treatment with phosphodiesterase, this light product was converted to the FMN form. The absorption spectrum of the FMN form has a peak at 464 nm, a shoulder at 450 nm in the visible region, and two peaks at 260 and 298 nm in the UV. Irradiation of free 6-azido-3-methyllumiflavin in the presence of a saturating concentration of oxygen yielded a light product whose absorbance and fluorescence spectra were very similar to those of the light product extracted from the enzyme, suggesting that the two had undergone some common photochemical change at the same place in the isoalloxazine ring. Analysis of the light product of 6-azido-3-methyllumiflavin with 1H NMR and FAB mass spectrometry suggested its possible structure with a new five-membered ring, C(6) = N-O-CH = C(7), adjacent to the benzene ring of the flavin.

Anaerobiosis↗
PubMed1992

Treatment of metastatic chemodectoma.

Six patients were treated for metastatic chemodectoma at Memorial Sloan-Kettering Cancer Center from 1971 through 1988. Four patients' primary tumors arose in the cervical region, and two arose in the retroperitoneum. Four patients received a total of eight different chemotherapeutic regimens, including cisplatin, doxorubicin, cyclophosphamide, and dacarbazine. Metastatic sites treated included bone, liver, lung, and retroperitoneum. No patient had a response to chemotherapy. Four patients received a total of nine courses of radiation therapy for palliation of bone metastases. Pain relief was complete in eight patients and partial in one. One patient was irradiated for a mass in the left psoas muscle, with stabilization of disease for 6 months after treatment. One patient was irradiated for epidural compression at T6, with resolution of neurologic symptoms and 50% clearing of the spinal block on follow-up myelogram. Recurrence or progression of disease in a previously irradiated site occurred in one patient 2 years after treatment. One patient was lost to follow-up 3 months after radiation therapy for epidural compression. The other five patients died of widespread metastatic disease 6 months to 9 years after initial treatment for their metastatic disease.

Adult↗
PubMed1991

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗