Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.
The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.
Meshalkina
L
L
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Nilsson
U
U
Wikner
C
C
Kostikowa
T
T
Schneider
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D004563:Electrochemistry; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism*
D014174:Transketolase; D013835:Thiamine Pyrophosphate