Mechanistic implications of mutations to the active site lysine of porphobilinogen syntheses

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of B-aminolevulinic ac id (ALA). The only characterized intermediate in the PBGS-catalyzed reactio n is a Schiff base that forms between the first ALA that binds and a conser ved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252, In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, an d K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. C-13 NMR studies of [3,5-C-13]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift d ifferences, i.e. environmental alterations. Mammalian PBGS is established t o have four functional active sites, whereas the crystal structure of E. co li PBGS shows eight spatially distinct and structurally equivalent subunits , Biochemical data for E. coli PBGS have been interpreted to support both f our and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change th at results in asymmetry. Product binding studies with wild-type E, coli PBG S and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, forma tion of the lysine to ALA Schiff base is not required to initiate the asymm etry that results in half-site reactivity.