The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides.
The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity. Like other transketolases, R. sphaeroides CbbT was found to be inactivated in the presence of oxygen. At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM. Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase. To investigate the role of cysteine 160 found in the active site of R. sphaeroides CbbT, this residue was targeted for mutagenesis. Cys160 was changed to alanine, serine, aspartate, and glutamate. To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity. Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding. The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue. In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.
Bobst
Cedric E
CE
The Ohio State Biochemistry Program, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.
Tabita
F Robert
FR
D000388:Air; D019943:Amino Acid Substitution; D000645:Ammonium Sulfate / Q000494:pharmacology; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001128:Argon / Q000494:pharmacology; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010427:Pentose Phosphate Pathway / Q000502:physiology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014174:Transketolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001051:Apoenzymes; D011994:Recombinant Proteins; D001128:Argon; D014174:Transketolase; D003545:Cysteine; D013835:Thiamine Pyrophosphate; D000645:Ammonium Sulfate