Reconstitution of holotransketolase is by a thiamin-diphosphate-magnesium complex.
When human erythrocyte apo-transketolase is mixed with cofactors and substrates, the progress curve exhibits a lag phase. Elimination of the lag phase requires the presence of saturating concentrations of cofactors, thiamin diphosphate and Mg2+. The most simple explanation of the observed hysteretic transition is that the slow binding of a Mg(2+)-thiamin-diphosphate species precedes slow isomerisation of the enzyme to the active form. Although the hysteretic transition involves more than one process, it does not involve the dissociation-association of enzyme subunits. The best estimate of the apparent Km, 1.59 +/- 0.23 microM, for the binding of Mg(2+)-thiamin diphosphate to transketolase was obtained in the presence of a high non-inhibitory concentration of magnesium and varied concentrations of thiamin diphosphate. Thus the reconstitution of the human enzyme differs from the yeast enzyme, which undergoes a rate-limiting dimerisation during reconstitution.
Booth
C K
CK
Department of Biochemistry, University of Queensland, Australia.
Nixon
P F
PF
D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism; D014174:Transketolase / Q000737:chemistry* / Q000378:metabolism
D014174:Transketolase; D008274:Magnesium; D013835:Thiamine Pyrophosphate