THE ALLOSTERIC REGULATION OF PYRUVATE-KINASE BY FRUCTOSE-1,6-BISPHOSPHATE

Background: Yeast pyruvate kinase (PK) catalyzes the final step in gly colysis, The enzyme therefore represents an important control point an d is allosterically activated by fructose-1,6-bisphosphate (FBP). In m ammals the enzyme is found as four different isozymes with different r egulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proli feration of certain cell types, as demonstrated by the expression of a n allosterically regulated isozyme in tumor cells. A model for the all osteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. Results: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in t he presence and absence of bound FBP. The allosteric site is located 4 0 Angstrom from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric act ivator is created by residues encoded by a region of the gene correspo nding to the alternately spliced exon of mammalian isozymes. FBP activ ation appears to induce several conformational changes among active-si te sidechains through a mechanism that is most likely to involve signi ficant domain motions, as previously hypothesized, Conclusions: The st ructure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes, The conformational differences observed betw een the active sites of inactive and fully active PK enzymes is in agr eement with the recently determined thermodynamic mechanism of alloste ric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.