Nonhydrolyzable ATP analogs

Figure 19-12 (A) Stereoscopic views of computer-assisted reconstructions of images of myosin heads attached to an F-actin filament centered between two thick filaments. Atomic structures of actin (Fig. 7-10) and of myosin heads (Fig. 19-15) have been built into the reconstructed images obtained by electron microscopy. (A) With the nonhydrolyzable ATP analog ATPPNP bound in the active sites. (B) Rigor. Two myosin heads are apparently bound to a single actin filament in (A). If they belong to the same myosin molecule the two C-terminal ends must be pulled together from the location shown here. In (B) a third head is attached, presumably from another myosin rod. This configuration is often seen in rigor. From Winkler et al.13i Courtesy of K. A. Taylor.

After ATP-G-actIn monomers are Incorporated Into a filament, the bound ATP Is slowly hydrolyzed to ADP. As a result of this hydrolysis, most of the filament consists of ADP-F-actin, but some ATP-F-actIn Is found at one end (see next subsection). However, ATP hydrolysis Is not essential for polymerization to take place, as evidenced by the ability of G-actin containing ADP or a nonhydrolyzable ATP analog to polymerize into filaments. 

Type III enzymes require ATP and Mg for ENase activity, but do not hydrolyze ATP. ATP behaves as an allosteric effector. The apparent fC for ATP (of coP15) is 7 JU.M (2). The optimal concentration of Mg is 5 mM. Nonhydrolyz-able ATP analogs can substitute for ATP in the ENase reaction, albeit at slower rates (3). ENase activity does not require AdoMet, but is stimulated by it (4—6). 

For many enzymes an ATP binding site has been revealed by study of nonhydrolyzable analogs of ATP such as "AMP-PNP" whose structure is shown in Fig. 12-31. AMP-PNP has been used in thousands of investigations of ATP-dependent processes. ... 

COPII vesicles were first recognized when cell-free extracts of yeast rough ER membranes were incubated with cytosol, ATP, and a nonhydrolyzable analog of GTP. The vesicles that formed from the ER membranes had a distinct coat, similar to that on COPI vesicles but composed of different proteins, designated COPII proteins. Yeast cells with mutations in the genes for COPII proteins are class B sec mutants and accumulate proteins In the rough ER (see Figure 17-5). Analysis of such mutants has revealed several proteins required for formation of COPII vesicles. 

COPI vesicles were first discovered when isolated Golgi fractions were incubated in a solution containing ATP, cytosol, and a nonhydrolyzable analog of GTP (see Figure 17-10). Subsequent analysis of these vesicles showed that the coat is formed from large cytosolic complexes, called coatomers, composed of seven polypeptide subunits. Yeast cells containing temperature-sensitive mutations in COPI proteins accumulate proteins in the... 

When researchers Incubated vesicles, axonal cytosol, and microtubules In the presence of AMPPNP, a nonhydrolyzable analog of ATP, the vesicles bound tightly to the microtubules... 

A resolution). AMPPNP (5 -adenylyl imidodiphosphate) is a nonhydrolyzable analog of ATP, which traps the transporter in the closed state. The apo state of the transporter is defined by a V-shaped chamber open to the cytoplasm and the inner leaflet of the membrane, with the two NBDs separated by about 5 nm (Fig. 4a). Addition of AMPPNP induces a tight packing of the NBDs which sandwiches the two nucleotides and induces an opening of the TMDs towards the outer leaflet of the bilayer. 

Imidophosphates -10 +0.9 Nonhydrolyzable analogs of ATP (or GTP) with p,y bridging linkage substituted (Tran EMnh and Roux, 1977). 

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