In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the
pyruvate kinase M(2)
enzyme and two of its missense mutations, H391Y and K422R, found in cells from
Bloom syndrome patients, prone to develop
cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y
mutant proteins maintained their homotetrameric structure, similar to the wild-type
protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate
phosphoenolpyruvate and behaved like a non-allosteric
protein with compromised cooperative binding. However, the affinity for
phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved
amino acid residues in long-range communications between the subunits of multimeric
proteins, the altered behavior of mutants is suggestive of their probable role in
tumor-promoting growth and metabolism in
Bloom syndrome patients with defective
pyruvate kinase M(2).