A TRANSKETOLASE ASSEMBLY DEFECT IN A WERNICKE-KORSAKOFF-SYNDROME PATIENT

Thiamine deficiency, a frequent complication of alcoholism, contribute s significantly to the development of damage in Various organ systems, including the brain. The molecular mechanisms that underlie the diffe rential vulnerabilities to thiamine deficiency of tissue and cell type s and among individuals ape not understood, investigations into these mechanisms have examined potential variations in thiamine utilizing en zymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. in the present study, w e examined a his-tagged human transketolase that was produced in and p urified from Escherichia coti cells. Previous findings demonstrated th at purified his-transketolase had a K-m app for cofactor and a thiamin e pyrophosphate-dependent lag period for attaining steady-state kineti cs that was similar to transketolase purified from human tissues, inte restingly, the time of the lag period, which is normally independent o f enzyme concentration, was found herein to be dependent on the concen tration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration- independent state required a cytosolic factor(s) derived from human ce lls. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced s ensitivity to thiamine deficiency.