Enzyme Memory

Abstract

When wheat germ hexokinase LI is rapidly mixed with its substrates in a stopped‐flow apparatus, the reaction proceeds and exhibits a ‘slow’ burst. The length of the negative induction time, τ, is independent of enzyme concentration but depends on glucose and magnesium‐ATP concentration. At low and high glucose concentrations, the burst disappears but becomes maximum at intermediate hexose concentrations. The approach to steady state can be considered and treated as a relaxation spectrum. Two relaxations can be detected experimentally: a ‘fast’ (0.2 s) and a ‘slow’ process (100 s). The ‘slow’ relaxation, studied at low glucose concentration, is very similar to the free enzyme relaxation which takes part in the mnemonical transition. This relaxation has been studied in further detail. When the reciprocal of the ‘slow’ relaxation time, λ 2 , is plotted against glucose concentration, one obtains a curve concave down, that reaches a plateau for high glucose concentrations. This result is taken to mean that glucose binding and enzyme transconformation occur as two distinct steps and that the binding step is faster than the transconformation process. The burst obtained at the start of the reaction is interpreted as indicative of a progressive shift from an enzyme species able to bind glucose easily and to undergo the proper conformation change, to an other form less able to bind glucose in a productive way. This shift would not be spontaneous but would be forced by the reaction process itself until the dynamic equilibrium that occurs under steady state is reached. Evidence in favor of enzyme memory has been obtained from pre‐mixing experiments with glucose 6‐phosphate. If the enzyme, already premixed with glucose 6‐phosphate in one of the drive syringes of the stopped‐flow apparatus, is mixed with the others reactants, the reaction exhibits a ‘slow’ lag instead of a burst. This transient would correspond to the shift from a weakly ‘reactive’ enzyme form to a strongly ‘reactive’ one. Thus, as expected with the concept of mnemonical transition, the free enzyme must occur under two forms having different reactivities for glucose and glucose 6‐phosphate.