Nucleotide Binding Domain

AAA family members have a nucleotide binding site reminiscent of Walker-type ATPases.52 In the S4 subfamily ofATPases the glycine-rich P-loop is always gly-pro-x-gly-x-gly-lys-thr (Box A) and the Mg2+ binding motif (Box B) almost always has the sequence asp-glu-ile-asp (Fig. 7.2). Nucleotidase activity has been reported for at least three RC ATPases, S4, S7, and S8.58-62 The Km for ATP hydrolysis by a GST-S4 fusion has been estimated at 5 ^M with a Vmax of approximately 7 pmoles/min/^g protein.58,59 By contrast, Makino et al determined the Km for ATP hydrolysis by rat S8 at ~35 ^M.62 This group also measured a Vmax for ATP hydrolysis similar to that of the yeast and human S4. The three ATPases, S4, S7, and S8, hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphates and inorganic phosphate. Unlike S4 and S8, whose nucleotide specificity resembles that of the rabbit reticulocyte regulatory and 26 S proteasome complexes, S7 exhibits a marked preference for ATP.60,63 Interestingly, two proteins that bind the retinoblastoma tumor suppressor protein (Rb) have been shown to alter the ATPase activity of S4 and S7. The human papillomavirus E7 oncoprotein binds S4 in vitro and stimulates its ATPase activity.59 By contrast, the centrosomal protein HEC down-regulates the ATPase activity of S7 upon binding and inhibits the degradation of cyclin B by the 26 S proteasome in vitro.60 The biological relevance of these results is unclear. On one hand, HEC preferentially binds free S7 (not RC or 26 S proteasome) and on the other, the stimulation of the activity of S4 by E7 is too modest to impact significantly the ATPase activity of the whole regulatory complex determined by Hoffman and Rechsteiner.63 In any event, the significance of these observations is an important question

Fig. 7.3. Binding of35S-labeled ATPases, S4 chimeras, and truncated forms ofS4 to RC subunits separated by SDS-PAGE. Regulatory complex ATPases, truncated forms of S4, and S4 chimeras were used to probe RC subunits bound to nitrocellulose filters as described in reference 33.

Fig. 7.3. Binding of35S-labeled ATPases, S4 chimeras, and truncated forms ofS4 to RC subunits separated by SDS-PAGE. Regulatory complex ATPases, truncated forms of S4, and S4 chimeras were used to probe RC subunits bound to nitrocellulose filters as described in reference 33.

that deserves further investigation since they suggest novel ways of modulating the activity of the 26 S proteasome during the cell cycle.

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