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V Massey

Publications and source records attributed to V Massey.

At least 181 records · Page 10Linked to original sources
PubMed1982

Evidence that the greening ligand in native butyryl-CoA dehydrogenase is a CoA persulfide.

Yellow butyryl-CoA dehydrogenase and general acyl-CoA dehydrogenase are "greened" by a mixture of coenzyme A plus elemental sulfur. The resultant stable complex contains an identical ligand with that present in native green butyryl-CoA dehydrogenase and has the same broad absorption band centered at 710 nm. Evidence is presented that the greening ligand is a CoA persulfide, possibly a mimic of the substrate carbanion thought to be generated early in the normal catalytic cycle. Variation in the position of the long wavelength band on replacement of FAD by a series of analogs of differing oxidation-reduction potential is consistent with a charge-transfer complex between a persulfide as the donor and oxidized flavin as the acceptor. The possible physiological and metabolic significance is discussed.

Acyl-CoA Dehydrogenases↗
PubMed1982

Steady-state kinetic studies on D-lactate dehydrogenase from Megasphera elsdenii.

Initial rate measurements were made of the oxidation of D-lactate and D-alpha-hydroxybutyrate by oxygen and potassium ferricyanide, catalyzed by D-lactate dehydrogenase from Megasphera elsdenii. The detailed kinetic work indicates a "ternary complex" type mechanism, with a complex of keto acid and reduced enzyme reacting with the electron acceptor at pH 8. However, as the pH is lowered, the double-reciprocal plots become nonlinear, with a downward curvature. This seems to be due to negative interactions within the protein rather than to a complexity of the kinetic mechanism. The variation of initial rate parameters at pH 8 with temperature yields nonlinear Arrhenius plots with a greater activation energy above the break point than below. This type of behavior has been previously reported only for fumarase (Massey, 1953). Studies with deuterated D-lactate show only a small isotope effect on phi 0 and phi 1 (KM/Vmax for lactate) but a large effect on phi 2 (Km/Vmax for ferricyanide).

Bacterial Proteins↗
PubMed1982

Stopped-flow kinetic studies on the D-lactate dehydrogenase from megasphera elsdenii.

The reaction of oxidized D-lactate dehydrogenase with D-lactate and reduced D-lactate dehydrogenase with pyruvate and oxygen was studied in a stopped-flow spectrophotometer. Oxidized enzyme was reduced in a triphasic manner by D-lactate at a variety of pH values. The fastest phase is thought to represent charge-transfer complex formation and is equal to an increase in long wavelength (510-700 nm) absorbance. The two slower phases seem to be due to flavin reduction. At pH 6 the proportion of the flavin reduction which proceeds by either phase is dependent upon the concentration of D-lactate. The reaction between reduced enzyme and oxygen is a simple second-order reaction. However, the oxidation of reduced D-lactate dehydrogenase by pyruvate is biphasic at pH 6 and monophasic at pH 8. At pH 8 there is a rapid increase in absorbance above 510 nm which is again ascribed to formation of a charge-transfer complex. These results are taken to indicate that D-lactate dehydrogenase may exist in two active conformations. At high pH the enzyme operates mainly through only one form while at low pH both forms participate in catalysis.

Hydrogen-Ion Concentration↗
PubMed1982

Metal binding to D-lactate dehydrogenase.

The kinetic and spectral properties of native and cobalt-substituted D-lactate dehydrogenase have been compared. Optical and MCD spectra are consistent with high-spin tetrahedral cobalt. The cobalt enzyme is strongly inhibited by D-lactate when oxygen is the electron acceptor. This is probably due to the formation of an abortive complex of reduced enzyme and lactate. Like the native zinc enzyme, the cobalt-substituted enzyme is reduced by substrate in a triphasic fashion involving a transient charge-transfer complex. The kinetics of the cobalt enzyme suggest that the metal is involved in substrate binding and reduction. A second metal-binding site is also described. Evidence is presented to suggest that the flavin cofactor and an essential thiol are involved in this site.

Binding Sites↗
PubMed1982

Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene.

D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.

Amino Acid Sequence↗
PubMed1982

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase.

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.

Amino Acids, Sulfur↗
PubMed1981

Studies on the oxidative half-reaction of xanthine oxidase.

The oxidative half-reaction of xanthine oxidase is reexamined with regard to the generation of the superoxide anion. By using cytochrome c reduction to monitor superoxide, it is found that the stoichiometry of superoxide produced to enzyme reoxidized is 2:1, significantly greater than previously reported (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4350-4362). Furthermore, the kinetics of superoxide-dependent cytochrome c reduction exhibits a pronounced lag during the rapid phase of enzyme reoxidation and a limiting rate identical with that of the slow phase of enzyme reoxidation. This indicates that superoxide is generated only in the last steps of the sequential removal of reducing equivalents from the enzyme by molecular oxygen. Experiments with the two-electron-reduced enzyme indicate that it too produces two superoxide ions for each molecule of enzyme reoxidized, demonstrating that it is the last two electrons to leave the enzyme in the course of reoxidation that form superoxide. The sequential scheme for the oxidative half-reaction must therefore be 6 leads to 4 leads to 2 leads to 1 leads to 0, where the numbers reflect the number of reducing equivalents in each intermediate. Using this scheme, both enzyme reoxidation and cytochrome c reduction can be accurately simulated. Reasons for the different behavior of the two-electron-reduced xanthine oxidase compared to the six- and four-electron-reduced enzyme are discussed.

Animals↗
PubMed1981

Active site probes of flavoproteins. Determination of the solvent accessibility of the flavin position 8 for a series of flavoproteins.

The chemical reactivity of 8-chloroflavins and 8-mercaptoflavins has been exploited in order to examine the orientation of protein-bound flavins relative to solvent. The apoprotein form of a series of flavoproteins was prepared and the native flavin was replaced by either 8-Cl-flavin or 8-mercaptoflavin (FAD, FMN, or riboflavin form as was appropriate). The reconstituted proteins were exposed to reagents capable of reacting with the group at position 8. The 8-Cl-proteins were challenged with sodium sulfide and thiophenol, while the 8-mercaptoproteins were faced with iodoacetamide and iodoacetic acid. The kinetics of the ensuing reactions served as a measure of the solvent availability of position 8 for the protein-bound flavin. These studies indicated that position 8 of flavin bound to melilotate hydroxylase, D-amino acid oxidase, old yellow enzyme, p-OH-benzoate hydroxylase, and flavodoxin is accessible to solvent, while position 8 on L-lactate oxidase, glucose oxidase, putrescine oxidase, and riboflavin-binding protein appears to be inaccessible. For luciferase, D-lactate dehydrogenase, and xanthine oxidase, the data suggest that position 8 is exposed but the results are inconclusive. The effect of ligand binding on the accessibility of position 8 was also studied. NADPH binding to 8-mercapto old yellow enzyme and benzoate binding to 8-Cl-D-amino acid oxidase results in complete blockage of previously available position 8. On the other hand, p-OH-benzoate hydroxylase and melilotate hydroxylase bind their respective substrates (p-OH-benzoate and melilotate) without significantly altering the reactivity of position 8.

Apoproteins↗
PubMed1981

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase.

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..

Animals↗
PubMed1980

Stabilization of lactate oxidase flavin anion radical by complex formation.

The red-colored flavin anion radical of lactate oxidase was formed by phtochemical reducion. the rdical was found to be reoxidized readily by molecular oxygen, with a rate constant of 4 x 10(5) M-1 min-1 at pH 7.0, 25 degrees C. On mixing the radical under anaerobic conditions with pyruvate, a change in spectrum typical of charge transfer interaction was found. The complex was composed of 1 eq of pyruvate per eq of enzyme flavin radical, with a Kd of 1.4 x 10(-5) M. The complex was sufficiently stable at 0 degrees C that it could be isolated free of exces keto acid by gel filtration under aerobic conditions. The isolted complex appears to be quite inert to oxidation by O2; the slow reoxidation which was observed was due to the slow dissociation of the complex and the subsequent fast reaction of O2 with the radical not in complex. The observed rate of oxidation is markedly dependent on temperature, with an activation energy of 25 kcal per mol.

Anaerobiosis↗