Transketolase kinetics. The slow reconstitution of the holoenzyme is due to rate-limiting dimerization of the subunits.

At pH 7.6 the monomeric apoenzyme is reactivated slowly when divalent metallic cation, (M2") and thiamin-PP are added, and a lag is seen in the progress curves in the enzyme assay.The apparent Km for thiamin-PP has two values, both of which differ, depending upon whether they are determined from the steady state velocity or from v-[ for the lag, the rate constant for transition between the initial and final steady state velocities.r-' is variable and depends upon the length of preincubation of apoenzyme with thiamin-PP and M2+, as well as on the particular M2`that is used.Further, at high concentrations of thiamin-PP r- reaches a constant, limiting value, which itself depends on the concentration of the enzyme and on the particular M2, that is present; r goes to zero at high concen- trations of thiamin-PP only when the ketose substrate is also present.r-1 increases linearly with [monomer]l7, suggesting that dimerization contributes sig- nificantly to the observed lag phase.Hill coefficients obtained from a wide range of thiamin-PP concentra- tions in the presence of Mg2e or Ca21 show that there is negative cooperativity in the binding of the coenzyme to the apoenzyme.Similar results were obtained with pyruvate decarboxylase at pH 6.2 except that <-did not vary with the enzyme concentration; this suggests that an isomerization, rather than an oligomerization, is the rate limiting step that causes the lag for this enzyme under these conditions.Possible mechanisms for the binding of thiamin-PP and the influence of the substrate on the observed kinetics are discussed for both of these enzymes.A characteristic common to all thiamin-PP-requiring en- zymes studied to date is the presence of a lag in the reaction