STRUCTURAL AND FUNCTIONAL-ANALYSIS OF PYRUVATE-KINASE FROM CORYNEBACTERIUM-GLUTAMICUM

Pyruvate kinase activity is an important element in the flux control o f the intermediate metabolism. The purified enzyme from Corynebacteriu m glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentrati on at the half-maximum rate (S-0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S-0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reacti on rate was 702 U/mg, which corresponded to an apparent k(cat) of 2,54 0 s(-1). The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutam icum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of th e native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequ ences of other bacterial pyruvate kinases showed 39 to 44% homology, w ith some regions being very strongly conserved.