The seperate interaction of the substrate fructose 1,6 bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving the alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of 7 to 8 at 24 MHz, demonstrating similar metal ion environments. The activator Co(III) EDTA did not affect Mn2+ binding to the neutral enzyme. In the presence of (α + β)methyl D fructofuranoside 1,6 bisphosphate, however, two sets, each containing four Mn2+ binding sites, were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6 bisphosphate concentration. A pre equilibrium sequential pathway model involving Mn2+ enzyme and the Mn2+ fructose 1,6 bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+ fructose 1,6 bisphosphate complex bound to the Mn2+ enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1975|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology