Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine. A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS). The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants. The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158. All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate. The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants. This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes. The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.