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 active sites, each with a non-covalently bound thiamine diphosphate and magnesium. Both subunits contribute residues at each site which are involved in cofactor binding and in catalysis. His-tagged transketolase, produced in E. coli, was similar to transketolase purified from human tissues with respect to Km apps for cofactor and substrates and with respect to cofactor-dependent hysteresis. Mutation of aspartate 155, corresponding to a conserved aspartate residue among thiamine diphosphate-binding proteins, resulted in an inactive protein which could not bind the cofactor-magnesium complex and which could not dimerize. The results are consistent with the suggestion that aspartate 155 is an important coordination site for magnesium. In support of this interpretation, binding of cofactor by wild type apo-transketolase required the presence of magnesium. Additionally, monomeric apo-his-transketolase required both magnesium and cofactor binding for dimer formation.
Wang
J J
JJ
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
Martin
P R
PR
Singleton
C K
CK
D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014174:Transketolase; D008274:Magnesium; D013835:Thiamine Pyrophosphate