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

Abstract

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

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

“…90% for the D-enantiomer. The stereochemistry was confirmed by stereospecific reaction of the pure enantiomers with L-lactate oxidase [14,15] and D-Iactate dehydrogenase [16,17].…”

“…The mixture had a fluorescence emission with maxima at 525 (identical to that of unmodified FMN) and 420 nm. The latter emission has an excitation spectrum with A max "" 340 nm, which presumably originated from the presence of several decay products of the modified FMN, as in the case of lactate oxidase [14,15]. Thus glycollate oxidase is apparently inactivated by a mechanism 247 similar to that operative with L-lactate oxidase, where a cyclic 4a,5-adduct of the enzyme-bound flavin is formed initially [13,14].…”

“…For the P. sativum enzyme no details were given on the mechanism of inactivation or the nature of the modification [12]. a-Hydroxybutynoic acid has been shown to in-'\ activate L-lactate oxidase and D-Iactate dehydro-,J genase, among other a-hydroxy acid oxidases [14][15][16][17], via formation of covalent flavin adducts. The structures of the inactivation products were shown to reflect the L-or D-specificity of the enzyme.…”

Structure of the flavin adduct formed in the suicide reaction of .alpha.-hydroxybutynoate with D-lactate dehydrogenase

“…With monoamine oxidase, the substrate analogue propargylamine reacts with the flavocoenzyme to form a covalent N(5) adduct having a cyanine structure (Chuang et al, 1974;Gaertner et al, 1976;Maycock et al, 1976). Several hydroxyacid oxidases [ L-lactate oxidase, Ghisla et al (1976) and Schonbrunn et al (1976); D-and L-lactate dehydrogenases, Walsh et al (1 972); L-hydroxyacid oxidase, Cromartie & Walsh (1975); cytochrome bt, Lederer (1974); glycollate oxidase, Jewess et al ( I 975)J have been reported to be irreversibly inactivated by 2-hydroxy-3-ynoic acids. In the case of L-lactate oxidase, the mechanism of inactivation was found to occur through primary formation of a cyclic N(5)-C(4a) adduct of the inactivator to the flavin coenzyme Schonbrunn et al, 1976) which subsequently decays by hydrolysis of the pyrimidine ring.…”

Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by .alpha.-hydroxybutynoate

“…Since the keto acid derived from the oxidation of this ahydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the a-hydrogen of a H B is the reactive species which covalently adds to the flavin. covalent addition at position N(5) occurred (Maycock et al, 1976), and with the L-specific lactate oxidase, cyclic addition to N(5) and C(4a) of the flavin nucleus was found (Walsh et al, 1972;Ghisla et al, 1976;Schonbrunn et al, 1976). Since these positions of the flavin coenzyme are electrophilic in the oxidized state, an attack of an electrophilic allene is considered unlikely.…”