Interaction of thiamin diphosphate and thiamin thiazolone diphosphate with wheat germ pyruvate decarboxylase.
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.
D002262:Carboxy-Lyases / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011767:Pyruvate Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology*; D014908:Triticum / Q000201:enzymology
D007202:Indicators and Reagents; C014222:thiamine thiazolone pyrophosphate; D002262:Carboxy-Lyases; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate