Purification and Properties of an Enzyme in Human Blood and Rat Liver Microsomes Catalyzing the Formation and Hydrolysis of γ-Lactones

An enzyme hydrolyzing 4- to 8-carbon γ-lactones has been purified 12- to 15-fold from rat liver and from human plasma. In rat liver the enzyme is located in the microsomal fraction and may be solubilized by deoxycholate treatment and separated from microsomal B-esterase by ammonium sulfate fractionation. The effects of a series of inhibitors on γ-lactonase, B-esterase purified from rat liver microsomes, and plasma cholinesterase were compared. The lactonase was inhibited by 1 mm ethylenediaminetetraacetate and by 0.1 mm p-chloromercuribenzoate but was unaffected by sodium benzoate, NaF, neostigmine, eserine, and diisopropylfluorophosphate. The last compound inactivates completely both the B-esterase and the cholinesterase, which were unaffected by ethylenediaminetetraacetate and were effectively inhibited by neostigmine. Together with substrate selectivity, these distinctions in inhibition establish the three enzymes as separate entities. The enzymatic nature of the reaction is evidenced by inactivation upon brief heating to 90°, purification of the active protein, a biologic Q10 = 1.56, a zero order reaction in either direction, and pH activity curves. In addition, no lactonase activity was found in homogenates of rat brain, spleen, kidney, heart, and diaphragm, although rat serum and plasma do contain the enzyme. The γ-lactonase enzymes also catalyze lactonization of the corresponding γhydroxy acids. The pH optimum for this reaction is 6.0 as compared to 8.6 for the hydrolytic reaction. The stoichiometry of lactone-hydroxy acid interconversion catalyzed by γ-lactonase was established by gas chromatographic identification of synthesized valerolactone and by acid regeneration of enzymatically degraded valerolactone. The enzyme does not hydrolyze simple aliphatic esters, acetylcholine, sugar lactones, or substituted aliphatic lactones such as pantolactone or 3-hydroxy-4-butyrolactone.