Gsj. Rao et al., Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated, ARCH BIOCH, 365(2), 1999, pp. 335-343
The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically mod
ify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby lo
cking the enzyme into a less active T-state. This enzyme form has a maximum
velocity that is 10% that of the native enzyme in the direction of fructos
e g-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation
for the substrate fructose 6-phosphate (S-0.5 (F6P) = 19 mM and n(H) = 2.2
) at pH 6.8 and a hyperbolic saturation curve for MgATP with a K-m identica
l to that for the native enzyme. The allosteric effecters, fructose 2,6-bis
phosphate and AMP, do not affect the S-0.5 for F6P but produce a slight (1.
5- and 2-fold, respectively) V-type activation with K-a values (effector co
ncentration required for half-maximal activation) of 0.40 and 0.24 mM, resp
ectively. Their activating effects are additive and not synergistic. The ki
netic mechanism for the modified enzyme is steady-state-ordered with MgATP
as the first substrate and MgADP as the last product to be released from th
e enzyme surface. The decrease in V and V/K values for the reactants likely
results from a decrease in the equilibrium constant for the isomerization
of the E:MgATP binary complex, thus favoring an unisomerized form. The V an
d V/K-F6P are pH dependent with similar pK values of about 7 on the acid si
de and 9.8 on the basic side. The microenvironment of the active site appea
rs to be affected minimally as evidenced by the similarity of the pK values
for the groups involved in the binding site for F6P in the modified and na
tive enzymes. (C) 1999 Academic Press.