Metabolism of the cysteine S-conjugate of busulfan involves a β-lyase reaction

The present work documents the first example of an enzymecatalyzed -elimination of a thioether from a sulfonium cysteine S-conjugate. -(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic -elimination reaction at pH 7.4 and 37°C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5 -phosphate (PLP). A PLPdependent enzyme in rat liver cytosol that catalyzes a -lyase reaction with THT-A was identified as cystathionine -lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway. Busulfan is a bifunctional alkylating agent used for the treatment of hematological and other malignancies before stem cell transplantation (e.g., Iwamoto et al., 2004). Busulfan is converted to a glutathione S-conjugate [L-glutamyl-(S-tetrahydrothiophenium)-L-alanylglycine] by direct interaction with glutathione (Ritter et al., 1999) and enzymatic catalysis by glutathione S-transferases, especially glutathione S-transferase A1–1 (Czerwinski et al., 1996; Gibbs et al., 1996; Ritter et al., 1999, 2002). The busulfan-glutathione adduct undergoes a base-catalyzed -elimination reaction yielding tetrahydrothiophene (Roberts and Warwick, 1961). Oxidation products of tetrahydrothiophene make up the majority of identified busulfan metabolites, although an enzymatic pathway leading to tetrahydrothiophene formation has not been elucidated. A hypothetical pathway to tetrahydrothiophene from busulfan could involve intermediates in the mercapturate pathway that convert glutathione S-conjugates to cysteine S-conjugates via the action of -glutamyltransferase and dipeptidase/cysteinylglycinase (Meister, 1989). The mercapturate pathway of busulfan metabolism was shown to occur in rats by the detection of the sulfonium mercapturate, N-acetyl-(S-tetrahydrothiophenium)-L-alanine in the urine (Hassan and Ehrsson, 1987). Therefore, it is probable that the busulfanglutathione adduct is converted in vivo to the corresponding cysteine S-conjugate [ -(S-tetrahydrothiophenium)-L-alanine, THT-A] by enzymes of the mercapturate pathway (Fig. 1). Theoretically, THT-A may also be formed nonenzymatically by the condensation of busulfan and cysteine. Because THT-A contains a good leaving group (nucleofuge), it is reasonable to predict a facile -elimination reaction to yield pyruvate, ammonium, and tetrahydrothiophene. Although this reaction is likely to occur slowly nonenzymatically, we hypothesize that one or more cysteine S-conjugate -lyases will accelerate the reaction. These lyases are pyridoxal 5 -phosphate (PLP)-dependent enzymes that catalyze -elimination reactions with L-cysteine S-conjugates [RSCH2CH(NH3 )CO2 ]. The enzyme-catalyzed reaction generates an RSH fragment and aminoacrylate [CH2 CH(NH3 )CO2 ]. The aminoacrylate is an enamine that rapidly tautomerizes to the more stable ketimine tautomer, -iminopropionate [CH3CH( NH2 )CO2 ], followed by hydrolysis to pyruvate [CH3C(O)CO2 ] and ammonium. The net reaction is shown in eq. 1 [see Cooper and Pinto (2006) for a recent review]. RSCH2CH NH3 CO2 H2O3 CH3C O CO2 NH4 RSH