NMR studies of fructose-1,6-bisphosphate aldolase from E. coli

Previous NMR studies of intact E. coli showed that during steady state anaerobic catabolism of glucose the main glycolytic intermediate detectable in these cells is fructose-1,6-bisphosphate (FBP), levels of which remain constant while the levels of glucose, lactate and succinate vary considerably. Upon feeding these cells glucose labeled with 13C at the C1 or C6 position, the level of scrambling of label between the C1 and C6 positions of FBP was low suggesting that the FBP-aldolase reaction is far from equilibrium. In order to account for these observations, a study was undertaken on FBP-aldolase from this organism. This enzyme is a dimeric Zn++ metalloenzyme with a M/sub r/ of 80,000. It was purified in gram quantities from an overproducer strain and was characterized by standard biochemical techniques prior to the NMR studies. 13C NMR experiments were conducted using [2-13C]dihydroxyacetone phosphate (DHAP) and [2,5-13C]fructose-1,6-biphosphate (FBP). Since these substrates can exist in solution in a number of interconvertible forms, the initial experiments determined the relative amounts of these forms and the rates of their interconversion. Subsequently, NMR experiments with the purified enzyme were conducted. Based upon these results, the author concludes that in E. coli the FBP-alkolase reaction appears to be the rate limiting step of anaerobic glycolysis