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The cleavage of the donor substrate D-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy. The enzymes are able to catalyze the cleavage of donor substrates, the first halfreaction, even in the absence of any acceptor substrate The pentose phosphate pathway is a major metabolic pathway in all cells. It consists of a dehydrogenase-decarboxylating system that converts D-glucose 6-phosphate to D-ribulose 5-phosphate, generating NADPHϩH ϩ for use in reductive biosynthesis, an isomerizing system that interconverts D-ribulose