Tryptophan Synthase Mutations That Alter Cofactor Chemistry Lead to Mechanism-Based Inactivation

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase β subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417−11422]. We now report that the S377D, S377E, and S377A β2 subunits form α2β2 complexes with the α subunit and activate the α subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent K d for dissociation of the α and β subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant α2β2 complexes exhibit very low activities in β elimination and β replacement reactions catalyzed at the β site in the presence of Na+, the activities and spectroscopic properties of the S377A α2β2 complex are partially repaired by addition of Cs+. The S377D and S377E α2β2 complexes, unlike the wild-type and S377A α2β2 complexes and the mutant β2 subunits, undergo irreversible substrate-induced inactivation by l-serine or by β-chloro-l-alanine. The rates of inactivation (k inact) are similar to the rates of catalysis (k cat). The partition ratios are very low (k cat/k inact = 0.25−3) and are affected by α subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the β subunit may promote the mechanism-based inactivation.