Biomedical subjects

V Massey

Publications and source records attributed to V Massey.

At least 91 records · Page 5Linked to original sources

PubMed1993

Redox potentials of milk xanthine dehydrogenase. Room temperature measurement of the FAD and 2Fe/2S center potentials.

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH), two forms of the same enzyme isolated from cow's milk, have differing redox potentials of their chromophores. Both XDH and XO are capable of accepting 8 electrons per active site cluster of redox acceptors. By titrating XDH with redox indicator dyes of various potentials, the potentials have been determined for the flavin as well as for the 2Fe/2S centers of the enzyme at pH 7.5, 25 degrees C. The redox potential for the FAD/FADH. half-potential was found to be -270 +/- 5 mV and that for the FADH./FADH2 half potential, -410 +/- 5 mV. The first flavin half potential is close to the value which has been reported for XO (Porras, A. G., and Palmer, G. (1982) J. Biol. Chem. 257, 11617-11626). However, the second FAD half-potential is 180 mV lower in XDH than in XO, creating a 140-mV separation between the FAD potentials in XDH. This separation gives rise to a maximum development of the flavin semiquinone in XDH near 0.9 equivalent as confirmed by EPR quantitation of FADH. formed during reductive titrations. The potentials of both the 2Fe/2S centers in XDH were determined and found to be identical to the values which were found for the iron-sulfur centers in XO.

Animals↗

PubMed1993

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Animals↗

PubMed1993

Role of tyrosine 129 in the active site of spinach glycolate oxidase.

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.

Alcohol Oxidoreductases↗

PubMed1993

Old Yellow Enzyme. The discovery of multiple isozymes and a family of related proteins.

Using fast protein liquid chromatography, we have separated native Old Yellow Enzyme from Brewer's Bottom Yeast into three distinct fractions. Two of these fractions are homodimeric forms of the enzyme while the third is the corresponding heterodimeric form. One of these homodimeric fractions is identical in every respect to OYE1, originally cloned from Brewer's Bottom Yeast (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). We have cloned, sequenced, and expressed a second Old Yellow Enzyme gene from Saccharomyces cerevisiae, showing close similarity, but not identity, with OYE1. Native Old Yellow Enzyme samples were also affinity-purified from a strain of S. cerevisiae and an OYE deletion mutant constructed from it. A total of at least seven isozymes of Old Yellow Enzyme have been discovered, each having slightly different characteristics ranging from surface charge to NADPH dehydrogenase activities with different electron acceptors, as well as N-terminal amino acid sequence. In addition, both recombinant enzymes showed considerable similarity to two proteins in the GenBank/EMBL data bank, a 60,000-dalton bile acid-inducible polypeptide in Eubacterium sp. (Mallonee, D. H., White, W. B., and Hylemon, P. B. (1990) J. Lipid Res. 172, 7011-7019) and a 72,000-dalton NADH oxidase in Thermoanaerobium brockii.

Amino Acid Sequence↗

PubMed1993

On the reaction mechanism of phenol hydroxylase. New information obtained by correlation of fluorescence and absorbance stopped flow studies.

Steps in the hydroxylation pathway of the flavoprotein phenol hydroxylase with resorcinol as substrate have been studied by a combination of fluorescence and absorbance stopped flow techniques. In the presence of azide, a series of highly fluorescent oxygenated flavin intermediates has been observed, corresponding to those previously detected by absorbance measurements (Detmer, K., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). In addition, yet another intermediate has been found as the immediate product of the reaction of the reduced enzyme with O2. This new species is non-fluorescent in the presence of azide, but fluorescent in the absence of monovalent anions and had escaped detection in previous absorbance studies because of the similarity in its rates of formation and conversion to the next intermediate and similarity in their spectra. These two early intermediates are tentatively identified as the anionic and protonated species of the flavin C4a-hydroperoxide or, alternatively, as two conformationally different forms of the enzyme hydroperoxide. The next intermediate, previously referred to as intermediate II, is also highly fluorescent and so is considered unlikely to be due to a complex of a flavin alkoxyl radical and a substituted cyclohexadienyl radical, as proposed by Anderson et al. (Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1990) J. Biol. Chem. 265, 1952-1957). The conversion of intermediate II to the next intermediate, intermediate III (the C4a-hydroxyflavin), is characterized by a large substrate deuterium isotope effect in the 320-390 nm range, but not by fluorescence or by absorbance at wavelengths > 400 nm. This is ascribed to dissociation from the enzyme of a cyclohexadienone product, leaving the enzyme in its C4a-hydroxyflavin form. The latter eliminates H2O to re-form oxidized flavin, but in a competing reaction, in the presence of excess substrate, forms a very stable complex, which decays orders of magnitude more slowly than the uncomplexed enzyme.

Azides↗

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↗

PubMed1991

The cloning and expression of a gene encoding Old Yellow Enzyme from Saccharomyces carlsbergensis.

We have identified a gene that encodes Old Yellow Enzyme in brewer's bottom yeast. The open reading frame encodes a polypeptide of 400 amino acids with Mr = 45,021. Using the T7 RNA polymerase system, recombinant enzyme was expressed in Escherichia coli. 17 mg of Old Yellow Enzyme was obtained from a 3-liter cell culture, and the recombinant enzyme had NADPH oxidase activity. On fast protein liquid chromatography separation, the recombinant enzyme showed a single large peak, while native enzyme from brewer's bottom yeast separated into five fractions on fast protein liquid chromatography. Southern blot analysis showed that there are at least two Old Yellow Enzyme genes in brewer's bottom yeast genomic DNA. These results suggest that the heterogeneity of Old Yellow Enzyme in Saccharomyces carlsbergensis is due to the presence of multiple genes.

Amino Acid Sequence↗

PubMed1991

The kinetic behavior of xanthine oxidase containing chemically modified flavins.

The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.

Flavins↗

PubMed1991

Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants.

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1991

Interpretation of the spectra observed during oxidation of p-hydroxybenzoate hydroxylase reconstituted with modified flavins.

Oxidation of reduced p-hydroxybenzoate hydroxylase by oxygen in the presence of 2,4-dihydroxybenzoate and azide proceeds via three well established intermediates. Reconstitution of the apoprotein with either 8-thiophenyl-FAD, 8-fluoro-FAD, 8-chloro-FAD, or 8-sulfonyl-FAD does not alter this sequence of events. However, the peak positions of the intermediate spectra are somewhat shifted relative to those of native enzyme. Comparison of the spectra for intermediates II and III leads to the conclusion that the spectrum for intermediate II is a composite. One component is the spectrum of an intermediate III-like species, and the other appears to be related to the substrate. The substrate component is pH-dependent, having an absorbance maximum of 386 nm (extinction, approximately 6,000 M-1 cm-1) at pH 6.6 which shifts to approximately 430 nm (extinction, approximately 11-13,000 M-1 cm-1) at pH 9.2, with a pK of 7.9. The pH dependence for the spectrum of the substrate component combined with the pH independence of the intermediate III-like spectrum satisfactorily accounts for the pH dependence observed for intermediate II, including the fact that the high pH spectrum of native intermediate II is qualitatively quite different from that of 8-sulfonyl-FAD intermediate II.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1991

Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues.

FAD-modified human glutathione reductases were reconstituted from apoenzyme using the FAD analogues 6-SH-FAD, 6-SCN-FAD, 6-OH-FAD, 6-NH2-FAD and 8-OH-FAD. The catalytic activities of the modified enzymes were substantially lower than for the native enzyme. All five species could be crystallized, but only those containing 6-SH-FAD, 6-OH-FAD and 6-NH2-FAD yielded crystals that could be analyzed. X-ray analyses and structural refinements were performed at 0.27 nm and 0.30 nm resolution resulting in R factors around 13.5%. The crystal structures showed the additional non-hydrogen atoms and small conformational changes of the polypeptide that were obviously induced by the substituents of the FAD analogues. The observed changes together with spectroscopic and activity data permit some conclusions about the chemical nature of the substituents.

Catalysis↗

PubMed1991

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.

Alcohol Oxidoreductases↗

PubMed1991

6-Mercapto-FAD and 6-thiocyanato-FAD as active site probes of phenol hydroxylase.

Recently, the synthesis and properties of several 6-substituted flavins as active site probes for flavoproteins have been reported (Ghisla, S., Massey, V., and Yagi, K. (1986) Biochemistry 25, 3282-3289). Here, we report results of experiments in which 6-thiocyanato-FAD and 6-mercapto-FAD have been substituted for the native flavin of phenol hydroxylase. The 6-SCN-FAD enzyme was converted spontaneously to the 6-mercaptoflavin form probably due to dissociation of flavin, followed by attack of external protein thiols. The pK alpha values of uncomplexed and phenol-bound 6-mercapto-FAD enzyme were determined. Both the spontaneously formed 6-mercapto-FAD enzyme and the enzyme reconstituted with preformed 6-mercapto-FAD were treated with a variety of thiol-specific reagents, and reaction rates were followed by spectroscopic means. Comparison with the corresponding rates found with free flavin suggested a high degree of accessibility to the flavin 6-position. Accessibility was somewhat decreased in the presence of phenol. Upon treatment with low concentrations of methyl methanethiosulfonate or N-ethylmaleimide (NEM), extremely rapid spectral changes were apparent. The former reaction, however, was reversed spontaneously within 2 h. Reaction with NEM was biphasic, with spectral changes consistent with the mechanism previously proposed (Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824), followed by a small absorbance decrease due to protein conformational changes. The NEM reaction is unusual, being easily reversed by addition of excess dithiothreitol.

Binding Sites↗