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Biomedical subjects

V Massey

Publications and source records attributed to V Massey.

At least 235 records · Page 13Linked to original sources
PubMed1976

The structure of the covalent flavin adduct formed between lactate oxidase and the suicide substrate 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoic acid is a suicide substrate for Mycobacterium smegmatis lactate oxidase. Inactivation occurs by covalent modification of enzyme-bound FMN and does not involve labeling of the apoprotein. The spectrum of the enzyme bound adduct suggests that it is a 4a, 5-dihydroflavin derivative. When this adduct is released from the enzyme, a complex mixture of unstable compounds is obtained. When the initially formed enzyme-bound adduct is reduced with NaBH4, a major stable species can be resolved from the enzyme and can be isolated and purified. The structure was established by appropriate isotope substitutions. Fourier transform NMR spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure II, Scheme II. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme II.

Binding Sites↗
PubMed1975

Identification of p-hydroxybenzaldehyde as the ligand in the green form of old yellow enzyme.

We have previously reported the isolation of old yellow enzyme complexed with a ligand of low molecular weight which imparts a distinctive charge-transfer absorption to the enzyme, making it green in color (Matthews, R. G., and Massey, V. (1969) J. Biol. Chem. 244, 1779-1789). This ligand has now been identified as p-hydroxybenzaldehyde by removal from the enzyme and characterization of its optical spectrum and subsequent mass spectral analysis. Similar compounds which are also bound to old yellow enzyme have been isolated from yeast extract and identified. These compounds give rise to complexes with old yellow enzyme which are characterized by broad long wavelength absorption bands, and they lead to inhibition of the NADPH-O2 oxidoreductase activity catalyzed by old yellow enzyme.

Benzaldehydes↗
PubMed1975

Kinetic and mechanistic studies on the reaction of melilotate hydroxylase with deuterated melilotate.

Melilotate has been synthesized from melilotate by iodiantion followed by reductive deiodination in the presence of deuterated hydrazine. The deuterated melilotate has been employed in investigations of the reaction mechanism of melilotate hydroxylase. Stopped-flow spectrophotometry has revealed no isotope effect in the formation or decay of the oxygenated intermediate which is observed when reduced melilotate hydroxylase reacts with molecular oxygen. Steady-state analysis has corroborated this result, and in addition shows that there is no isotope effect in the reductive cycle of the enzyme mechanism. This analysis does reveal a reproducible 8% decrease in Vmax for the enzyme when using deuterated melilotate. These observations are compatible with the thesis that the above intermediate is an oxygenated form of the reduced flavine prosthetic group and that the last step of the proposed mechanism is rapid and involves a primary isotope effect. The existence of the NIH shift mechanism has been studied using combined gas chromatography-mass spectrometry. No evidence could be obtained for intramolecular migration of deuterium during the hydroxylation reaction. However, the small amount of migration expected when phenols are hydroxylated precludes elimination of the NIH shift as a possibility.

Chromatography, Gas↗
PubMed1975

Mechanism of inactivation of the flavoenzyme lactate oxidase by oxalate.

Lactate oxidase from Mycobacterium smegmatic is strongly inhibited by oxalate. The inhibition is reversible in the absence and irreversible in the presence of light. Oxalate is bound to the enzyme in a two-step process with an over-all Kd of 1.6 times 10--5 M. The first step is a fast second order reaction with k-1/k1 equals 8.3 times 10--3 M, leading to a Complex I. This complex is then reversibly converted to a different Complex II in a slow first order reaction (k2 equals 40 min--1; k-2 equals 0.07 min--1), which is accompanied by major spectral perturbations of the flavin spectrum. With oxamate, two steps could not be demonstrated, and its binding is described by a single step reversible process, which is second order in oxamate (k1 equals 6.8 times 10-3 M--1 s--1, k-1 equals 28 s--1). Upon illumination the enzyme-oxalate Complex II is converted very rapidly at position N(5) of the flavin. Slow hydrolysis in the dark under anaerobic conditions subsequently yields free reduced enzyme. The light reaction of the oxamate comples is, in contrast, very slow and yields a stable N(5) urea adduct of the reduced flavin.

Binding Sites↗