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

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...

1. We studied toluene metabolism in dog liver microsomes and the major metabolite was benzyl alcohol with o- and p-cresol as minor metabolites. 2. The enzyme kinetics of toluene biotransformation were examined by means of Lineweaver-Burk analyses. The Michaelis-Menten values differed among the three pathways, the order being; Km, o-cresol > p-cresol > benzyl alcohol; Vmax, benzyl alcohol > o-cresol > p-cresol; and Cl(int), benzyl alcohol > p-cresol > o-cresol. 3. The formation of benzyl alcohol, o- and p-cresol from toluene was substantially inhibited by the P4502E inhibitors such as DDC (diethyldithiocarbamate) and 4-methylpyrazole in all pathways, with IC50's in the range of 0.02-0.59 mM. The P4502B inhibitors, metyrapone and secobarbital also inhibited benzyl alcohol and p-cresol formation, whereas o-cresol was not inhibited by these latter compounds. 4. Anti-rat P4502E1 antibodies inhibited benzyl alcohol, o- and p-cresol formation from 26 to 30% 0.2 ml serum/mg microsomal protein. Furthermore, anti-rat P4502B1/2 antibody inhibited benzyl alcohol and p-cresol formation (47 and 44% respectively), but not that of o-cresol. Anti-rat P4502C11/6 antibody also inhibited benzyl alcohol and p-cresol formation 31 and 24% respectively in a similar manner to that by the anti-rat P4502B1/2 antibody. 5. These results suggested that the P4502B, 2C and 2E isozymes in dog liver contribute to the formation of benzyl alcohol and p-cresol from toluene, and 2E isozyme preferentially contributes to the formation of o-cresol.