The allosteric regulation of pyruvate kinase - A site-directed mutagenesisstudy

Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathw ay. The regulatory properties of Escherichia coli mere investigated by muta ting six charged residues involved in interdomain salt bridges (Arg(271), A rg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activ ator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the inte rface between A and C domains within one subunit, The R271L and K413Q mutan t enzymes exhibit altered kinetic properties. In K413Q, there is partial en zyme activation, whereas R271L is characterized by a bias toward the T-stat e in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inact ive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganiz ation of the interface with the creation of a network of interactions simil ar to that observed in the crystal structures of R-state yeast and M1 PK pr oteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1,6-bisphosphate, the allosteric activ ator. R431E exhibits only slight changes in the regulatory properties. Conv ersely, K382Q displays a highly altered responsiveness to the activator, su ggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion tha t domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the ge ometry of the fructose 1,6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused b y mutations in human erythrocyte PK gene.