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 (1.2 mM) and for substrate inhibition by 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 CBS-catalyzed 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), = 0.083 mM). The (catR)/ versus pH profile of ytCBS is bell-shaped with 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 microM, respectively.