Effect of transketolase substrates on holoenzyme reconstitution and stability.
The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.
Esakova
O A
OA
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
Khanova
E A
EA
Meshalkina
L E
LE
Golbik
R
R
Hübner
G
G
Kochetov
G A
GA
D002118:Calcium / Q000737:chemistry; D004795:Enzyme Stability / Q000502:physiology; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014174:Transketolase / Q000737:chemistry* / Q000378:metabolism*
D020035:Holoenzymes; D009943:Organophosphorus Compounds; D014174:Transketolase; D008274:Magnesium; D013835:Thiamine Pyrophosphate; D002118:Calcium