Rapid-quench and isotope-trapping studies on fructose-1,6-diphosphatase

Abstract

Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of 4 identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a 2nd reaction phase liberating additional Pi that completes the initial turnover of 2 or 4 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 4 subunits turn over in the pre-steady-state; with Mn2+ only 2 of the 4 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 2 the 4 subunits now remain active with Mg2+, as well as with Mn2+ in the pre-steady state. So that the amount of Pi released can be accounted for, a .beta. .fwdarw. .alpha. anomerization or direct .beta. utilization is required at the active site of one subunit. Such behavior is consistent with the 2-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for .alpha. and .beta. substrate analogs. Under these limiting conditions the subsequent steady-state rate is limited by the .beta. .fwdarw. .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.