Kinetics of the yeast cystathionine β-synthase forward and reverse reactions:: Continuous assays and the equilibrium constant for the reaction

Cystathionine beta-synthase (CBS) is a pyridoxal-phosphate-dependent enzyme that catalyzes a beta-replacement reaction in which the hydroxyl group of serine (L-Ser) is displaced by the thiol of homocysteine (L-Hcys) to form cystathionine (L-Cth) in the first step of the trans-sulfuration pathway. A new continuous assay for the forward reaction, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, is described. It alleviates product inhibition by L-Cth and revealed that the values for K-m(L-Ser) (1.2 mM) and for substrate inhibition by L-Hcys (K-iFl(L-Hcys) = 2.0 mM) are lower than those previously reported. A continuous, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB)-based assay for the CBScatalyzed hydrolysis Of L-Cth to L-Ser and L-Hcys provides a tool for investigation of the reverse reaction (k(catR) = 0.56 s(-1), K-m(L-Cth) = 0.083 mM). The k(catR)/K-m(L-Cth) versus pH profile of ytCBS is bell-shaped with m M a pH optimum of 8.3, and the pK(a) values for the acidic and basic limbs are 8.05 and 8.63, respectively. The latter is assigned to the alpha-amino group of L-Cth (pK(a) = 8.54). The internal aldimine of ytCBS remains protonated at pH < 11; therefore, the acidic pK(a) is assigned to an enzyme functionality that is not associated with the internal aldimine. K-eq was determined directly and from the kinetic parameters, and the values are 0.61 and 1.2 muM, respectively.