Isotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum

Gibson, Grant E., Isotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum. Doctor of Philosophy (Biomedical Sciences), May, 1996, 91 pages, 2 tables, 18 figures, 2 schemes, 1 reaction, 2 mechanisms, 1 diagram, bibliography, 61 titles. The natives Ascaris suum 6-phosphofructo-1-kinase (nPFK) and a chemically modified form (dPFK) which is desensitized to allosteric behavior have been studied using isotope partitioning and initial velocity techniques to determine the kinetic mechanism as well as the effects of fructose 2,6-biphosphate (F26P2) and Mg2+ on the mechanism. At 8 mM Mg2+, complete trapping (Pmax≈100%) of E:MgATP complex as fructose 1-(32P), 6-biphosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism ((Rao, G.S.J., Harris, B.G., and Cook, P.F. (1987) J. Biol. Chem. 262, 14074-14079) with MgATP binding before fructose 6-phosphate (F6P). A saturating amount of F26P2 causes no change in the trapping parameters for nPFK but causes a decrease in both Pmax and K’F6P for dPFK. The partial trapping of E:MgATP in the presence of F26P2 for dPFK at high MG2+ suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initiial velocity studies at 8 mM Mg2+ confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the dPFK: (14C)F6P complex could be detected 8mM Mg2+, indicating either that dPFK:14C-F6P complex does not form or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. Initial velocity data indicate that a second Mg2+ ion in addition to the one bound in MgATP is an essential activator of Ascaris suum PFK which decreases the off-rate for MgATP. Kact for Mg2+ is estimated to be 0.47±0.08mM. Isotope partitioning data at 0.1 mM Mg2+ indicate that dPFK is able to trap only 20% of the E:MgATP* both in the presence and absence of F26P2, consistent with a faster off-rate for MgATP at low Mg2+ than at high Mg2+. Partial trapping of MgATP* at low Mg2+ again suggests a random binding of substrates. Noncompetitive Ara5P inhibition versus MgATP at low Mg2+ confirms the random mechanism. An active site role both in binding MgATP and in facilitating catalysis is proposed for the second Mg2+. Furthermore, calculations from the isotope partitioning and initial velocity data as well as changes that are seen in the circular dichroic spectra for both nPFK and dPFK indicate that an enzyme structural isomerization occurs upon binding mgATP.