The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.

The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca 2؉ -dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Transketolase (TKT 3 ; EC 2.2.1.1) is a ubiquitous enzyme in cellular carbon metabolism and requires thiamine diphosphate (ThDP), the biologically active derivative of vitamin B1, and Ca 2ϩ ions as cofactors for enzymatic activity (1, 2). TKT catalyzes the reversible transfer of two-carbon (1,2-dihydroxyethyl) units from ketose phosphates to the C1 position of aldose phosphates and thus provides, together with the Schiff base-forming transaldolase, a reversible link between glycolysis and the pentose phosphate pathway. This shunt permits cells a flexible adaptation to different metabolic needs as the pentose phosphate pathway supplies intermediates for other metabolic pathways; generates precursors for biosynthesis of nucleotides, aromatic amino acids, and vitamins; and further produces NADPH for sustaining the glutathione level and for reductive biosynthetic pathways of, for example, cholesterol and fatty acids.TKT acts on different ketose phosphate (donor) and aldose phosphate (acceptor) substrates of variable carbon chain length (3-7 carbons) in two major, essentially reversible reactions. REACTIONS 1 AND 2A simplified reaction scheme for the TKT-catalyzed conversion of substrates X5P and R5P into products S7P and G3P is shown in Fig. 1. The reaction cycle can be subdivided into a donor half-reaction (donor ligation and cleavage) and an acceptor half-reaction (acceptor ligation and product liberation) (3).After formation of the reactive ylide form of ThDP, the C2 carbanion of ThDP attacks the carbonyl of donor X5P in a nucleophilic manner to yield the covalent donor-ThDP adduct X5P-ThDP (step 1). Ionization of C3-OH and cleavage of the scissile C2-C3 bond of X5P-ThDP results in the formation of product G3P and of the 1,2-dihydroxylethyl-ThDP (DHEThDP) carbanion/enamine intermediate (step 2). This intermediate may then react with either G3P (reverse reaction of step 2) or R5P in competing equilibria. In the latter case, C2␣ of DHE-ThDP ligates to C1 of R5P (in the acyclic form), yielding * This work was supported by a grant from the "Fonds der Chemischen Industrie" (stipend to S. L.) and the Deutsche Forschungsgemeinschaft-funded Gö ttingen Graduate School for Neurosciences and Molecular Biosciences (to K. T.

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