ADP-vanadate

Smith, C. A., Rayment, 1. X-ray structure of the magnesium (11). ADP-vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry 35 5404-5407, 1996. 

Figure 1.4 Low-resolution projection maps of P-gp obtained from 2Dc7Stals in the nucleotide-free state (a) and in the presence of nucleotides (AMP-PNP) (b) and ADP/vanadate (c).These2D maps suggest, but do not unequivocally demonstrate, major conformational changes associated with the binding of nucleotides. Major molecular rearrangement between the AMP-PNP-bound state and the nucleotide-free state was later confirmed by generating 3D structures...

Describe the large conformational difference between myosin-ADP and myosin-ATP (same as myosin-ADP-vanadate). Locate switch 1, switch 11, and the relay helix in relation to the P-loop, and explain how they cause the protein to flex. 

Ambudkar, S. V., Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates, J. Biol. Chem. 2001, 276, 8657-8664. 

This enzyme [EC 3.4.21.53], also known as endopepti-dase La, ATP-dependent serine proteinase, and ATP-dependent protease La, catalyzes the hydrolysis of peptide bonds in large proteins (for example, globin, casein, and denaturated serum albumin) in the presence of ATP (which is hydrolyzed to ADP and orthophosphate). Vanadate ion inhibits both reactions. A similar enzyme occurs in animal mitochondria. Protease La belongs to the peptidase family S16. 

As noted earlier, studies with inhibitors have been of great value. One mole of ouabain binds per enzyme complex and inhibits all enzyme functions. It provides a convenient marker for the extracellular surface of the enzyme. Oligomycin inhibits the (Na+, K+)-ATPase but not the K+-phosphatase reaction. It stimulates the ADP/ATP exchange reaction and this led to the postulate for two phosphoenzymes in the reaction scheme. Anomalous kinetic behaviour for (Na+, K+)-ATPase, over some years, was eventually recognized57 to be due to a vanadate impurity in ATP, which binds with high affinity to the low affinity ATP site and with low affinity to the high affinity ATP site. In accord with this, vanadate effectively inhibits the K+-phosphatase... 

The complex with ADP and aluminium fluoride is thought to resemble the early ADP.Pi state immediately after hydrolysis, whereas the complex with ADP and vanadate may represent a late state in which the phosphate (mimicked by vanadate) has moved quite a long distance (15 A) from the active center to the surface of the motor domain. There it is fixed by two hydrogen bonds to the solvent exposed tips of the switch-1 loop region (L9) at one side and the switch-2 loop (Lll) at the other side. 

Vanadate-induced photo-oxidation of serine and subsequent cleavage of the protein were also observed for ribulose-l,5-diphoshate carboxylase/oxygenase from spinach leaves. Activity could be restored by treatment with sodium boronate, which reduces formylglycine back to serine.Similarly, vanadate-induced UV oxidation of threonine (Thr353, at the phosphorylation site) in the sarcoplasmatic reticulum (SR) Ca +-ATPase can be restored by reduction with Na[BH4]. For monovanadate to become catalyti-cally active it is essential that Mg + and ADP bind to the phosphorylation site of the enzyme in its Ca +-receptive conformation, suggesting the formation of an active MgADPV as proposed for the myosin fragmentation. In contrast, decavanadate catalyses the photo-oxidation of a serine (Seri 86) residue in the SR ATPase in the absence of ADP.P bl... 

Like the spinach enzyme, the pea ATPase is activated equally by Mg2+ and Mn2+ and hydrolyzes a broad range of nucleoside triphosphates, but not ADP, AMP, or monophosphorylated substrates. Although pea chloroplast envelope membranes have ADPase and pyrophosphatase activity, we conclude that the activities are distinct from the ATPase activity. The envelope ATPase differs from putative transport ATPases characterized in other plant membranes in that it is not inhibited by vanadate or DCCD, nor is it stimulated by potassium. However, a role for this activity in proton efflux and ion transport cannot be ruled out, because the envelope vesicles may be sufficiently leaky that protons and ions can diffuse freely across the membrane. This might limit any stimulatory effect of K+ and uncouplers. Evidence supporting a role for the ATPase in proton transport will depend on further characterization of the envelope vesicles, and/or purification and reconstitution of the ATPase into artificial lipid vesicles. 

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