The dimeric molecular chaperone Hsp90 is required for the activation and stabilization of hundreds of substrate proteins, many of which participate in signal transduction pathways. The activation process depends on the hydrolysis of ATP by Hsp90. Hsp90 consists of a C-terminal dimerization domain, a middle domain, which may interact with substrate protein, and an N-terminal ATP-binding domain. A complex cycle of conformational changes has been proposed for the ATPase cycle of yeast Hsp90, where a critical step during the reaction requires the transient N-terminal dimerization of the two protomers. The ATPase cycle of human Hsp90 is less well understood, and significant differences have been proposed regarding key mechanistic aspects. ATP hydrolysis by human Hsp90␣ and Hsp90␤ is 10-fold slower than that of yeast Hsp90. Despite these differences, our experiments suggest that the underlying enzymatic mechanisms are highly similar. In both cases, a concerted conformational rearrangement involving the N-terminal domains of both subunits is controlling the rate of ATP turnover, and N-terminal cross-talk determines the rate-limiting steps. Furthermore, similar to yeast Hsp90, the slow ATP hydrolysis by human Hsp90s can be stimulated up to over 100-fold by the addition of the co-chaperone Aha1 from either human or yeast origin. Together, our results show that the basic principles of the Hsp90 ATPase reaction are conserved between yeast and humans, including the dimerization of the N-terminal domains and its regulation by the repositioning of the ATP lid from its original position to a catalytically competent one.

Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a k cat of 0.36 min

Hsp90 is an ATP-dependent molecular chaperone whose mechanism is not yet understood in detail. Here, we present the first ATPase cycle for the mitochondrial member of the Hsp90 family called Trap1 (tumor necrosis factor receptor-associated protein 1). Using biochemical, thermodynamic, and rapid kinetic methods we dissected the kinetics of the nucleotide-regulated rearrangements between the open and the closed conformations. Surprisingly, upon ATP binding, Trap1 shifts predominantly to the closed conformation (70%), but, unlike cytosolic Hsp90 from yeast, this process is rather slow at 0.076 s ؊1 . Because reopening (0.034 s ؊1 ) is about ten times faster than hydrolysis (k hyd ‫؍‬ 0.0039 s ؊1 ), which is the rate-limiting step, Trap1 is not able to commit ATP to hydrolysis. The proposed ATPase cycle was further scrutinized by a global fitting procedure that utilizes all relevant experimental data simultaneously. This analysis corroborates our model of a two-step binding mechanism of ATP followed by irreversible ATP hydrolysis and a one-step product (ADP) release. 90-kDa heat shock protein (Hsp90)3 is a molecular chaperone highly conserved from bacteria to mammals. The dependence on Hsp90 apparently reflects the relative complexity of the organism. For example, bacteria lacking HtpG (the prokaryotic homolog of mammalian Hsp90) appear to be fully viable (1), whereas disruption of Hsp90 in eukaryotic organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila, is lethal (2-5). The family of Hsp90-related molecular chaperones mediates protein folding, assembly, and stability of a variety of substrate proteins in vivo. In vitro, Hsp90 suppresses the aggregation of non-native proteins (6) and can also promote the refolding of substrates in cooperation with the Hsp70 system (7-10).In eukaryotic organisms, several members of the Hsp90 family can be found in different compartments such as the cytosol, endoplasmic reticulum, mitochondria and in chloroplasts (11). The organellar versions differ from their cytosolic counterparts in several aspects. The most striking is that they seem to lack specific co-chaperones. While Grp94, the Hsp90 homolog of the endoplasmic reticulum has evolved from its cytosolic counterpart, Trap1, the mitochondrial version, is a descendent of prokaryotic Hsp90 (11). Whether they share a general mechanism of ATP hydrolysis is not known. In this study, we focused on the biochemical and kinetic analysis of the Hsp90 homolog Trap1, which is localized in mitochondria (12-14). Originally, Trap1 was identified as a tumor necrosis factor receptor-associated protein (15). Trap1 is 31% identical to human Hsp90, 34% to Escherichia coli HtpG, 33% to yeast Hsp90, and 31% to Grp94. For the N-terminal ATP-binding domain alone, the identities are even higher. There are a few identified Trap1 substrates such as the retinoblastoma protein (16), the tumor suppressor EXT proteins (17) and Myc, which influences cell proliferation (18). Recently, an interaction partner of Trap1, which is locat...

The reaction of di(n-propyl)cyclopropenone ( 7), di(n-butyl)cyclopropenone ( 16), and bicyclo[12.1.0]pentadeca-1( 14)-en-7-yn-15-one (21) with CpCo(CO) 2 and CpCo(cod) yielded CpCo-complexed benzoquinone and cyclopentadienone derivatives. When 16 was reacted with CpCo(CO) 2 and a 10-fold surplus of 4-octyne, a mixture of CpCo-capped benzoquinone 25 and cyclopentadienone 26 was isolated. The reaction of cyclopropenone 16 containing a 13 C-labeled CO group with CpCo(CO) 2 yielded a CpCo-capped tetrakis(n-butyl)-p-benzoquinone with one 13 C nucleus per molecule as the main product. The mechanism of the formation of p-benzoquinone is discussed on the basis of the results of trapping and labeling experiments.