Isolation of citroylformic acid-gamma-lactone from a commerical batch of oxaloacetic acid: inhibition of apotyrosine aminotransferase conversion to holoenzyme by this substance.

Citroylformic acid-γ-lactone (CFA, 1-keto-2,4-dihydroxy-4-carboxyadipenoic acid(2–3)-1,4-lactone), isolated from a commercial batch of oxaloacetate, inhibited conversion of rat liver apotyrosine aminotransferase (EC 2.6.1.5) to holoenzyme. Using partially purified enzyme, the K_i was determined to be less than 0.7 mm. A more definitive K_i was difficult to obtain because at pH 7 CFA had a half-life of about 2 hr. Inhibition of the enzyme by CFA was stereospecific and reversible; the S (?) stereoisomer was approximately 10 times more inhibitory than its R(+) antipode, and over 90% of inhibited enzyme was recoverable after overnight dialysis. Preineubation of apotyrosine aminotransferase with its coenzyme (pyridoxal phosphate) prevented inhibition by CFA, and a substantial fraction of enzyme that had been inhibited by CFA could be readily reactivated by addition of high concentrations of pyridoxal phosphate. Studies with inhibitor analogs indicated that both a partially unsaturated lactone ring and a stereospecific carboxymethyl group are required for maximal inhibitory activity. The sodium salts of citroylformic acid and oxalopyruvic acid, formed by the hydrolysis of their respective lactones, were not inhibitory; 1-keto-2,4-dihydroxy-4-carboxyadipic acid-γ-lactone and little inhibitory activity, and 1-keto-2,4-dihydroxyglutarenoic acid-γ-lactone and 1-keto-2,4-dihydroxybutene-γ-lactone were somewhat better inhibitors than the R(+) stereoisomer of CFA. The possibility that CFA is a naturally occurring biological substance is discussed.