Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase.
Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [alpha-Fru-1,6-P2]/[FBPase] ratio. With Mg2+ in the presence of excess alpha-fructose 1,6-bisphosphate (alpha-Fru-1,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting alpha-anomer concentrations only two of the four subunits now remain active with Mg2+ as well as with Mn2+ in the pre steady state. However, so that the amount of Pi released can be accounted for, a beta leads to alpha anomerization or direct beta utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for alpha and beta substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the beta leads to alpha solution anomerization. These data in combination with pulse--chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+ is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.
D000818:Animals; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D007554:Isotopes; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008433:Mathematics; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors
D005635:Fructosediphosphates; D007554:Isotopes; D046911:Macromolecular Substances; D010710:Phosphates; D008345:Manganese; D006597:Fructose-Bisphosphatase; D008274:Magnesium; C029063:fructose-1,6-diphosphate