Thiamine deficiency, a frequent complication of
alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to
thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in
thiamine utilizing
enzymes.
Transketolase is a homodimeric
enzyme containing two molecules of noncovalently bound
thiamine pyrophosphate. In the present study, we examined a his-tagged human
transketolase that was produced in and purified from Escherichia coli cells. Previous findings demonstrated that purified his-
transketolase had a Km app for cofactor and a
thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to
transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of
enzyme concentration, was found herein to be dependent on the concentration 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 cells. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to
thiamine deficiency.