EXAMINATION OF SUBSTRATE-BINDING IN THIAMIN DIPHOSPHATE-DEPENDENT TRANSKETOLASE BY PROTEIN CRYSTALLOGRAPHY AND SITE-DIRECTED MUTAGENESIS

The three-dimensional structure of the quaternary complex of Saccharom yces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acce ptor substrate erythrose-4-phosphate has been determined to 2.4 Angstr om resolution by protein crystallographic methods. Erythrose-4-phospha te was generated by enzymatic cleavage of fructose-6-phosphate. The ov erall structure of the enzyme in the quaternary complex is very simila r to the structure of the holoenzyme; no large conformational changes upon substrate binding were found. The substrate binds in a deep cleft between the two subunits. The phosphate group of the substrate intera cts with the side chains of the conserved residues Arg(359), Arg(528), His(469), and Ser(386) at the entrance of this cleft. The aldehyde mo iety of the sugar phosphate is located in the vicinity of the C-2 carb on atom of the thiazolium ring of the cofactor. The aldehyde oxygen fo rms hydrogen bonds to the side chains of the residues His(30) and His( 263). One of the hydroxyl groups of the sugar phosphate forms a hydrog en bond to the side chain of Asp(477) The preference of the enzyme for donor substrates with D-three configuration at the C-3 and C-4 positi ons and for a hydroxylated acceptor substrates can be understood from the pattern of hydrogen bonds between enzyme and substrate. Amino acid replacements by site directed mutagenesis of residues Arg(359), Arg(5 28), and His(469) at the phosphate binding site yield mutant enzymes w ith considerable residual catalytic activity but increased K-m values for the donor and in particular acceptor substrate, consistent with a role for these residues in phosphate binding. Replacement of Asp(477) by alanine results in a mutant enzyme impaired in catalytic activity a nd with increased K-m values for donor and acceptor substrates. These findings suggest a role for this amino acid in substrate binding and c atalysis.