Properties of fructose-1,6-bisphosphate aldolase from Escherichia coli: an NMR analysis.
A class II Zn(2+)-dependent fructose-1,6-bisphosphate (FBP)- aldolase was purified from an overproducer strain of Escherichia coli and characterized by standard biochemical techniques and 13C NMR spectroscopy. The principal finding of these studies was identification, by 13C NMR spectroscopy, of an enzyme-bound reaction intermediate, the enediol(ate) form of dihydroxyacetone phosphate (DHAP). Formation of this intermediate requires the presence of Zn2+ and is pH dependent, with increasing amounts of this tautomer appearing at alkaline pH's. This pH dependence closely parallels the pH activity profile of the enzyme, suggesting an involvement of the enediol-DHAP form in the reaction pathway. In addition to these results the following observations were made on this enzyme: (a) E. coli FBP aldolase binds and utilizes only the carbonyl forms of FBP and DHAP; (b) the function of Zn2+ in this metalloaldolase appears to be polarization of the C = O bond of DHAP; (c) activity of this enzyme is unaffected by glycolytic intermediates or nucleotide phosphates such as ATP. Although these studies provide some information about the catalytic mechanism of E. coli FBP aldolase, they do not provide an explanation for the apparent regulation of this enzyme reported in previous in vivo NMR studies. While the possibility that the enzyme is allosterically regulated cannot be excluded at this time, an interesting possibility suggested by this and other studies is that in E. coli glycolytic substrates may be channeled through a multienzyme complex.
Szwergold
B S
BS
Department of NMR and Medical Spectroscopy, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Ugurbil
K
K
Brown
T R
TR
D001665:Binding Sites; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013379:Substrate Specificity; D015032:Zinc
D004492:Edetic Acid; D005634:Fructose-Bisphosphate Aldolase; D015032:Zinc