ASPARTATE-155 OF HUMAN TRANSKETOLASE IS ESSENTIAL FOR THIAMINE DIPHOSPHATE MAGNESIUM BINDING, AND COFACTOR BINDING IS REQUIRED FOR DIMER FORMATION

Active human transketolase is a homodimeric enzyme possessing two acti ve sites, each with a non-covalently bound thiamine diphosphate and ma gnesium. Both subunits contribute residues at each site which are invo lved in cofactor binding and in catalysis. His-tagged transketolase, p roduced in E. coli, was similar to transketolase purified from human t issues with respect to K-m apps for cofactor and substrates and with r espect to cofactor-dependent hysteresis. Mutation of aspartate 155, co rresponding to a conserved aspartate residue among thiamine diphosphat e-binding proteins, resulted in an inactive protein which could not bi nd the cofactor-magnesium complex and which could not dimerize. The re sults are consistent with the suggestion that aspartate 155 is an impo rtant coordination site for magnesium. In support of this interpretati on, binding of cofactor by wild type apo-transketolase required the pr esence of magnesium. Additionally, monomeric apo-his-transketolase req uired both magnesium and cofactor binding for dimer formation. (C) 199 7 Elsevier Science B.V.