CRITICAL ROLE OF ARG(433) IN RAT TRANSKETOLASE ACTIVITY AS PROBED BY SITE-DIRECTED MUTAGENESIS

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK ) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To id entify the critical arginine, four highly conserved arginine residues of rat TK (Arg(102), Arg(350), Arg(433) and Arg(506)) were replaced wi th alanine by site-directed mutagenesis. Wild-type and mutant TK prote ins were produced in Escherichia coli and characterized. The Arg(102) --> Ala mutant exhibited similar catalytic activity to the wild-type e nzyme, whereas Arg(350) --> Ala, Arg(506) --> Ala and Arg(433) --> Ala mutants exhibited 36.7, 37.0 and 6.1%, of the wild-type activity resp ectively. Three recombinant proteins (wild-type, Arg(350) --> Ala and Arg(433) --> Ala) were purified to apparent homogeneity using Ni2+-aff inity chromatography and further characterized. All these proteins wer e able to form homodimers (148 kDa), as shown by immunoblot analysis s ubsequent to non-denaturing gel electrophoresis. The Arg(433) --> Ala mutant protein was less stable than the wild-type and Arg(350) --> Ala proteins at 55 degrees C. Kinetic analyses revealed that both V-max a nd K-m values were markedly affected in the Arg(433) -->Ala mutant. Th e K-m values for two substrates xylulose 5-phosphate and ribose 5-phos phate were 11.5- and 24.3-fold higher respectively. The k(cat)/K-m val ues of the Arg(433) --> Ala mutant for the two substrates were less th an 1% of those of the wild-type protein. Molecular modelling of the ra t TK revealed that Arg(433) of one monomer has three potential hydroge n-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modellin g data suggest the critical role of the previously uncharacterized Arg (433) in TK activity.