MUTATION OF AN ACTIVE-SITE RESIDUE OF TRYPTOPHAN SYNTHASE (BETA-SERINE 377) ALTERS COFACTOR CHEMISTRY

To better understand how an enzyme controls cofactor chemistry, we hav e changed a tryptophan synthase residue that interacts with the pyridi ne nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (be ta-Ser(377)) to a negatively charged Asp or Glu, The spectroscopic pro perties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an A sp residue interacts with the pyridine nitrogen of pyridoxal phosphate . The absorption spectrum of each mutant enzyme undergoes a pH-depende nt change (pK(a) similar to 7.7) from a form with a protonated interna l aldimine nitrogen (lambda(max), = 416 nm) to a deprotonated form (la mbda(max), = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta(2), subunit and alpha(2) beta(2), complex are pH-independent. The reaction of the S377D alpha(2) beta(2) complex wi th L-serine, L-tryptophan, and other substrates results in the accumul ation of pronounced absorption bands (lambda(max), = 498-510 nm) ascri bed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonat ed pyridine nitrogen of pyridoxal phosphate, reducing the pK(a) of the internal aldimine nitrogen and promoting formation of quinonoid inter mediates.