Crystallization and properties of <scp>l</scp>-lactate oxidase from <i>Mycobacterium smegmatis</i>

Sullivan, Patrick A.

doi:10.1042/bj1100363

Cited by 46 publications

(35 citation statements)

References 13 publications

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“…The observation of a biphasic reaction between lactate oxidase and DL-2-hydroxy-3-butynoate leading to the formation of this long wavelength intermediate is not surprising. D isomers of ahydroxy acids are strong competitive inhibitors (Sullivan, 1968) and dissociate from lactate oxidase slowly (Massey and Ghisla, unpublished). Therefore, by analogy to the reaction of lactate oxidase with DL-lactate, the first rapid phase observed in Figure 3 probably corresponds to the reaction of the enzyme complexed with the L isomer of 2-hydroxy-3-butynoate.…”

Section: Resultsmentioning

confidence: 99%

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Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate]2-hydroxy-3-butynoate

Ghisla¹,

Ogata²,

Massey³

et al. 1976

Biochemistry

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2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, 0 2 . Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band ( A, , , 600 nm) and lack of fluorescence, making it easily distinguished from The use of acetylenic substrate analogues as specific irreversible inactivators of target enzymes was initiated by Bloch and his colleagues (Bloch, 1969), using 3-decynoyl thiolesters to inactivate /3-hydroxydecanoyl thiolester dehydrase. Specificity devolved from the fact that the enzyme converts the acetylenic molecule into a conjugated allenic thiolester, the actual alkylating agent, a t its active site. Subsequently, acetylenic analogues have been used to inactivate thiolase (Holland et al., 1973), plasma amine oxidase (Hevey et al., 1973), ketosteroid isomerase (Batzold and Robinson, 1 9 7 9 , and several pyridoxal phosphate-dependent enzymes (Abeles and Walsh, 1973;Marcotte and Walsh, 1975). In each instance carbanionic intermediates are suspected during catalysis, and covalent modification of some amino acid residue on the protein (apoprotein) is indicated.In addition several flavoenzymes can be induced to undergo irreversible inactivation by acetylenic analogues. Pargyline (N-benzyl-N-methylpropynylamine), long known to inhibit the flavin-linked monoamine oxidase (Hellerman and Erwin, 1968), appears to modify the FAD cofactor (Chuang et al., 1974). With N-dimethylpropynylamine as substrate, a covalent adduct forms a t N S of the flavin coenzyme (Maycock et al., 1976). In 1972 we reported the preliminary observation that the Mycobacterium smegmatis L-lactate oxidase is irreversibly inactivated by 2-hydroxy-3-butynoate due to covalent

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Section: Resultsmentioning

confidence: 99%

“…D~-2-Hydroxy-3-butynoic acid was prepared by published procedures (Leonard, 1956;Verny and Vessiere, 1967). FAD was obtained from Sigma and phosphodiesterase (Naja naja venom) from Ross Allen Reptile Institute, Silver Springs, Florida.…”

Section: Methodsmentioning

confidence: 99%

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate]2-hydroxy-3-butynoate

Ghisla¹,

Ogata²,

Massey³

et al. 1976

Biochemistry

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“…Lactate oxidase concentration was determined from the reported extinction coefficient of enzyme bound F M N a t 452 nm (Sullivan, 1968).…”

Section: Methodsmentioning

confidence: 99%

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

Schönbrunn¹,

Abeles²,

Walsh³

et al. 1976

Biochemistry

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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 L -l a c t a t e oxidase ( E C 1.13.12.4) from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of L-lactate.OHThe substrate analogue DL-2-hydroxy-3-butynoate (I) acts as both a substrate (i.e., is oxidatively decarboxylated) and an irreversible inactivator of the enzyme (Walsh et al., 1972a). OH HC=CCHCOO- IEvidence has been presented that 2-hydroxy-3-butynoate is bound a t the same binding site for both the inactivation and oxidation reactions . Which of the two processes occurs is determined by the concentration of the second substrate, oxygen (Walsh et al., 1972a;Ghisla et al., 1976). The lower the concentration of oxygen, the fewer turnovers the enzyme undergoes before it is inactivated. Under anaerobic conditions 1 mol of L-2-hydroxy-3-butynoate per mol of flavin containing active site causes complete inactivation (Walsh et al., 1972a). Both the @-hydroxyl group and the 0-acetylenic linkage of 2-hydroxy-3-butynoic acid are necessary for inactivation to occur since neither 3-butynoic acid nor vinylglycolic acid inactivate the enzyme (Walsh et al.. 1972a 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 N M R spectroscopy, chemical reactivity, and synthesis of a model compound. The structure of the isolated adduct is structure 11, Scheme 11. The structure proposed for the adduct initially formed on the enzyme is structure VII, Scheme 11. results in covalent modification of the flavin coenzyme but does not affect the apoprotein (Walsh et al., 1972a). The enzymebound flavin-2-hydroxy-3-butynoate adduct displays absorption peaks with maxima at 320 and 368 nm characteristic of 4a-mono-or 4a,5-disubstituted 4a,5-dihydroflavin derivatives (Ghisla et al., 1973;Ghisla et al., 1974). Previously reported experinients with DL-2-hydroxy-3-butynoate labeled with tritium at C2 and C4 have indicated that the C2 hydrogen of 2-hydroxy-3-butynoate is not present in the flavin adduct, while the C4 hydrogen is present, but no longer in an acetylenic linkage (Walsh et al., 1972a).As shown in the preceding paper ) the modified flavin of the inactivated enzyme is reduced by borohydride. Such reactivity, as well as the spectroscopic properties, suggested strongly that the flavin derivative formed on reaction of lactate oxidase with 2-hydroxy-3-butynoate is a cyclic adduct involving substitution both at the h'(5) and C(4a) positions of the flavin. In this paper we report experiments which establish the structure of the flavin...

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“…Comparison of glycollate oxidase from pea leaves and from other sources [1, 5,13] with related enzymes which oxidize L-lactate, e.g., with L-lactate oxidase from Mycobacteria [29][30][31] or with flavocytochrome b 2 from yeast [32]. reveals some interesting aspects: FMN is the coenzyme in most cases, and in particular when glycollate is the preferred substrate, suggesting a correlation of the two properties.…”

Section: Inactivation Of the Enzyme With A-hydroxybutynoic Acidmentioning

confidence: 99%

Studies on glycollate oxidase from pea leaves determination of stereospecificity and mode of inhibition by α-hydroxybutynoate

Fendrich¹,

Ghisla²

1982

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Crystallization and properties of l-lactate oxidase from Mycobacterium smegmatis

Cited by 46 publications

References 13 publications

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate]2-hydroxy-3-butynoate

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate]2-hydroxy-3-butynoate

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

Studies on glycollate oxidase from pea leaves determination of stereospecificity and mode of inhibition by α-hydroxybutynoate

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