The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is alloster ically inhibited by L-serine, the end product of its metabolic pathway. Pre vious results have shown that inhibition by serine has a large effect on V- max and only a small or negligible effect on K-m. PGDH is thus classified a s a V-type allosteric enzyme. In this study, the active site of PGDH has be en studied by site directed mutagenesis to assess the role of-certain resid ues in substrate binding and catalysis. These consist of a group of cationi c residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charge d substrate, as well as the only tryptophan residue found in PG;DH and whic h fits into a hydrophobic pocket immediately adjacent to the active site hi stidine residue. Interestingly Trp-139' and Lys-141' are part of the polype ptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mu tants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subuni ts.