The binding of MgADP to the E187A mutant of E-coli phosphofructokinase.

Phosphofructokinase (PFK) from E. coli is al los t eric ally activated by MgADP and inhibited by phosphoenolpyruvate (PEP). MgADP binds at both the allosteric and the active site, with the latter interaction antagonized by the binding of the substrate fructose-6-phosphate (F6P). X-ray crystallography indicates that glutamate at position 187 in the allosteric site binds Mg++. which also coordinates with ADP. Lau and Fersht (Nature 326 :811-812, 1987) first reported that mutating the glutamate to alanine (E187A) causes PEP to become an activator of F6P binding. MgADP binding to the E187A mutant at both the allosteric site and the active site can be detected by changes in the steady-state fluorescence emission and polarization of the intrinsic tryptophan. The effect of MgADP binding at the allosteric site on F6P affinity is negligible, although MgADP binding at the active site remains antagonistic to F6P binding. In addition, Mg ++ alone can bind to the enzyme even in the absence of the supposedly required charged residue. MgADP binds to the enzyme with a higher affinity (Kd = 8M) than either Mg ++ (Kd = 0.12mM) or ADP f Kd = 0.04 mM) alone. MeADP is shown to bind competitively with PEP at the allosteric site, as in the wild type enzyme.

The binding of MgADP to the E187A mutant of E-coli phosphofructokinase. Academic Article