Non-hydrolyzable ATP

In the Walker crystal structure of Fj-ATPase, the three non-catalytic a sites are liganded with the non-hydrolyzable ATP analog MgAMP-PNP. In contrast, the three catalytic (3 sites possess different conformations. One of the catalytic sites in the structure binds the analog MgAMP-PNP and is designated as Pjp another site binds MgADP and is denoted by (3dp, while the third site is empty and distorted and is called (3e [21]. In further contrast, the nucleotide-free subcomplex of ATP synthase is a symmetric trimer [36]. 

Non-hydrolyzable ATP analogs such as adenylyl imidodiphos-phate (AMP-PNP, Figure 2d) and B,Y-adenylyl methylene bisphospho-nate (CH.-ATP,Figure 2e) have been used as inhibitors of kinases because they can bind in a similar manner to the natural substrate... 

Hsp90 forms a stable complex with the co-chaperone p23/Sbal (Fang et al., 1998 Johnson and Toft, 1994 1995) in the presence of non-hydrolyzable ATP analogs, such as AMP-PNP. Such a stable complex can also be formed by incubation with ATP in the presence of molybdate ions in which a stable Hsp90—ADP—MoO - complex is progressively formed. Molybdate has long been included in Hsp90 buffers, prior to... 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

Fig. 29. Sketch of the three-dimensional structure of the bovine Fi-ATPase (left). Subunits labelled with the subscript DP have bound ADP, those with TP have bound AMP-PNP, a non-hydrolyzable analogue of ATP, while those with E have no associated nucleotide. The figure on the right shows only one a- and one p-subunit, corresponding to the two on the left by shading. "DELSEED segment contains amino-acids 394 to 400 in MF,. Figure source Abrahams, Leslie, Latter and Walker (1994) Structure at 2.8 A rBso/ution of FrATPase from bovine heart mitochondria. Nature 370 623.

This assay is used to measure adenylyl cyclase activity downstream of the receptor, by directly activating lysates with GTPyS - a non-hydrolyzable form of GTP - or MnSO - an ATP chelator that provides a measurement of the intrinsic adenylyl cyclase catalytic activity. MnSO measurements are therefore used as an alternative readout of AGA expression levels. 

To test the ability of ARF to enhance the ADP-ribosylation of simple guanidino compounds, its activity was examined in the NADragmatine ADP-ribosyltransferase assay (Table 1). ARF increased the toxin-catalyzed ADP-ribosylation of agmatine approximately 3-fold. As noted with the auto-ADP-ribosylation reaction, activation of ADP-ribosylagmatine formation was dependent on GTP or its non-hydrolyzable analogues. GDP, GDPpS, ATP and App(NH)p were consistently less effective. ARF also increased the toxin-catalyzed ADP-ribosylation of proteins unrelated to the... 

The non-muscle myosins share with muscle myosin the ability to hydrolyze ATP and to form cross links between actin filaments, but there is considerable variation in their other physical, chemical and enzymatic properties. This variation in the myosins may be related to the diversity of movements which different cells exhibit. 

Interestingly, if organisms contain non-Bi2 cobalt enzymes such as nitrilases (Koba-yashi and Shimizu 1998), an additional slow, chemisosmotically driven uptake system (NiCoT protein family) is co-expressed with the enzyme (Komeda et al. 1997). ATP-hydrolyzing uptake systems for cobalt (e.g., ABC-transport systems) are not known. This indicates that cobalt for B12-enzymes may indeed be imported as cobalamin, Co(II) for other enzymes by NiCoT transport systems and that Co(II)-import by other systems may not be important in the natural environment of the cells. Co(II) is of medium toxicity and is detoxified by efflux systems (CDF protein family, RND-driven CBA-export systems) in bacteria and yeasts (Nies 2003). 

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