Abstract
The interactions of the apoenzyme of wheat germ pyruvate decarboxylase with thiamin diphosphate and with thiamin thiazolone diphosphate have been investigated. The results test hypotheses concerning the structure of the transition state for decarboxylation of the enzyme-bound adduct of pyruvate and thiamin diphosphate. Thiamin thiazolone diphosphate, a possible transition state analogue, binds to the apoenzyme by a two-step process. The first is slow and reversible (k = 200 M-1 s-1; K = 5 X 10(-7) M). The second step is irreversible (k = 1 X 10(-6) s-1). The rate constant for activation by thiamin diphosphate is 160 M-1 s-1. Thiamin diphosphate is released very slowly from the holoenzyme (k = 2 X 10(-5) s-1). Thiamin thiazolone diphosphate competitively inhibits activation of the apoenzyme by thiamin diphosphate, Ki = 2 X 10(-6) M. Km for thiamin diphosphate is only 3 times larger. Thiamin thiazolone is solvated preferentially to thiamin in 2-butanol, a medium whose polarity should resemble that of the binding site. It is concluded that the observed high affinity of thiamin thiazolone diphosphate for the apoenzyme is the result of a combination of effects which do not require the assumption that it is an analogue of the transition state for the decarboxylation of enzyme-bound 2-(2-lactyl) thiamin diphosphate.
Highlights
The affinity of T T D P for wheat germ pyruvate decarboxylase has not beenreported
High affinity of thiamin thiazolone diphosphatefor the apoenzyme is the result of a combination of effects which do not require theassumption that it is an analogue of the transitionstate for the decarboxylationof
Compared to thiamin thiazolone is a factor of 9. It is clear if the rate of release of T T D P from the enzyme is used as a that thiamin has a very low affinity for the less polar solvent criterion for measurement of affinity, T T D P appears to bind in the presence of water
Summary
The affinity of T T D P for wheat germ pyruvate decarboxylase has not beenreported. Since loss of carbon dioxide fromLTDPisthe key step in T D P catalysis of reactions of pyruvate,itisimportanttounderstandthe sources of affinity for the active site. To clarify this situation, we have investigated the interactions of T T D P with wheat germ pyruvate decarboxylase. High affinity of thiamin thiazolone diphosphatefor the apoenzyme is the result of a combination of effects. NMR spectra were measured on Varian T-60 ('H) and Bruker WP-80 (3'P) instruments. The adduct undergoes loss of carbon dioxide and acquires a proton to form HETDP prior to thexpulsion of acetaldehyde or further reaction
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