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Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

ABSTRACT: 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 identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

SUBMITTER: Soh Y

PROVIDER: S-EPMC1219594 | biostudies-other | 1998 Jul

REPOSITORIES: biostudies-other

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Project description:Dephosphocoenzyme A kinase performs the transfer of the ?-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.

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Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

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