Nucleotides, hydrolysis

Both the 26S proteasome and the RC hydrolyze all four nucleotide triphosphates, with ATP and CTP preferred over GTP and UTP [58]. Although ATP hydrolysis is required for conjugate degradation, the two processes are not strictly coupled. Complete inhibition of the peptidase activity of the 26S proteasome by calpain inhibitor I has little effect on the ATPase activity of the enzyme. The nucleotidase activities of the RC and the 26S proteasome closely resemble those of E. coli Lon protease, which is composed of identical subunits that possess both proteolytic and nucleotidase activities in the same polypeptide chain. Like the regulatory complex and 26S proteasome, Lon hydrolyzes all four ribonucleotide triphosphates, but not ADP or AMP [18]. 

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Phalloidin and phallacidin are cyclic peptides from the mushroom Amanita phalloides that stabilize F-actin. Phalloidin binds to residues 114-118 of an actin protomere and blocks nucleotide exchange without interfering with nucleotide hydrolysis. It enhances the rate of nucleation as well as that of elongation. It slowly penetrates the cell membrane and is used for immunocytochemical localization of F-actin. 

Monomeric actin binds ATP very tightly with an association constant Ka of 1 O M in low ionic strength buffers in the presence of Ca ions. A polymerization cycle involves addition of the ATP-monomer to the polymer end, hydrolysis of ATP on the incorporated subunit, liberation of Pi in solution, and dissociation of the ADP-monomer. Exchange of ATP for bound ADP occurs on the monomer only, and precedes its involvement in another polymerization cycle. Therefore, monomer-polymer exchange reactions are linked to the expenditure of energy exactly one mol of ATP per mol of actin is incorporated into actin filaments. As a result, up to 40% of the ATP consumed in motile cells is used to maintain the dynamic state of actin. Thus, it is important to understand how the free energy of nucleotide hydrolysis is utilized in cytoskeleton assembly. 

Polymer growth J(c) showed nonlinear monomer concentration dependence in the presence of ATP (Carrier et al., 1984), while in the presence of ADP, the plot of J(c) versus monomer concentration for actin was a straight line, as expected for reversible polymerization. The data imply that newly incorporated subunits dissociate from the filament at a slower rate than internal ADP-subunits in other words, (a) the effect of nucleotide hydrolysis is to decrease the stability of the polymer by increasing k and (b) nucleotide hydrolysis is uncoupled from polymerization and occurs in a step that follows incorporation of a ATP-subunit in the polymer. Newly incorporated, slowly dissociating, terminal ATP-subunits form a stable cap at the ends of F-actin filaments. 

Hicks, B.W. and Parsons, S.M., 1992, Characterization of the P-type and V-type ATPases of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to acetylchohne transport. J. Neurochem., 58 1211-1220. 

As noted earlier, it is conceivable that the rate constants at each end are different however, Wegner noted that in the absence of nucleotide hydrolysis the assembly and disassembly reactions are in fact reversible. The rate constants are directly related to the equilibrium constants for protomer-polymer interactions at each end ... 

Furthermore, although the rates of addition and loss may differ at each end (i.e., the transition states for the individual association-dissociation reactions may depend on the polarity of protomer-polymer interactions), the initial state (protomer + MT ) and the final state (MT +i) are independent of the pathway for protomer addition. This result is true because addition or loss of a protomer at either end leads to structurally indistinguishable polymers. Thus, the energetics of the reactions at each end are the same, and Ki equals for the case of no nucleotide hydrolysis accompanying assembly. We may then reexpress the rate equations for protomer addition or loss to the two ends in the following manner ... 

Wegner also treated the case wherein assembly is coupled to nucleotide hydrolysis. Here, we consider a slight modification of his model to deal with the microtubule process. Normally, the concentration of GTP is maintained by use of a GTP-regenerating system (MacNeal et ai, 1977), and the system at the steady-state plateau of assembly can be described as in Scheme II. Under these conditions, the assembly-disassembly reactions are no longer reversible, and the primed rate constants are used to emphasize that we are dealing with a different case. The rate equations for the two ends are now given as ... 

Finally, the theory for the bioenergetics and kinetics of microtubule assembly and disassembly of microtubules has been extended by Hill and Kirschner (1983). They consider the coupling of nucleotide hydrolysis in terms of the energetics of the [GTP]/[GDP][PJ mass action ratio, the possible effects of force imparted by attachment of tubules to barriers on the rate constants, and other intriguing aspects of protomer-polymer exchange kinetics and thermodynamics. Unfortunately, much of their theory remains to be tested, and an evaluation of its importance in revealing the subtleties of assembly/disassembly remains for future investigations. 

Rodriguez, A.C. (2002) Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional latch in the mechatrism of reverse gyrase. J. Biol. Chem. 277, 29865-29873. 

Assembly Nucleotide Hydrolysis are NOT Coupled to Each Other... 

MICROTUBULE ASSEMBLY/DISASSEMBLY KINETICS. Cellular microtubules must undergo turnover, and nucleotide hydrolysis appears to play a central role in priming microtubules for their eventual disassembly. Two fundamentally different assembly/disassembly mechanisms persist during what has been termed steady-state polymerization both rely on GTP hydrolysis to provide a source of Gibbs free energy to sustain the steady-state condition . ... 

Berman, D. M., Kozasa, T., and Gilman, A. G. (1996a). The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. / Biol. Chem. 271, 27209-27212. 

Melki, R., Fievez, S., and Carlier, M.-F. (1996). Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mer-capto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay. Biochemistry 35, 12038-12045. 

Sonnenburg WK, Beavo JA (1994) Cyclic GMP and regulation of cyclic nucleotide hydrolysis. Adv Pharmacol 26 87-114... 

We hydrolyzed ATP and ADP in 1 N and 0.1 N HC1 and in buffered solutions at pH 4j nd 8 in which the hydrolysis medium was variously enriched in °0 to either 10% or 20%. To assess the isotopic enrichment of each such solution for use in the nucleotide hydrolysis experiments, we hydrolyzed PCI, in the solution, esterified the resultant phosphoric acid/inorganic phosphate (P.) by reaction with diazomethane, and determined the isotopic distribution of the trimethyl phosphate (TMPO) by mass spectrometry. The 1 N and 0.1 N HC1 hydrolyses were allowed to proceed for 45 min and 10 hr, respectively, at 70, insuring complete conversion of ATP into AMP + 2P. The pH 8 hydrolyses were allowed to proceed for 36 hr at 70 to a point (20-25% completion) at which the ratio of ADP to AMP established that 96% and 4%, respectively, of the P. released had arisen by the primary and secondary hydrolysis steps, namely, ATP ADP + P. and ADP " AMP + P. 0The pH 4 hydrolyses were allowed to proceed for 24 hr, also at 70, to 40% completion. 

Martin C, Berridge G, Mistry P, et al. Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis. Biochemistry 2000 39 (39) 11901—11906. 

DNA polymerase and 5 - 3 exonuclease functions of the enzyme. Nucleotide hydrolysis in the 5 - 3 direction concomitant with nucleotide polymerization results in translocation of the position of the discontinuity by a process termed nick translation (Fig. 2) (30). Discontinuities or nicks in DNA can be introduced into intact DNA by limited digestion with pancreatic DNase I, which generates 3 -hydroxyl termini in double-stranded DNA. If radioactive nucleotides are used in the reaction with DNA polymerase 1, randomly and uniformly labeled DNA is produced (31). 

Fig. 2. Strategies for uniform labeling of double-stranded DNA. (A) Nick translauon involves the 5 - 3 exonuclease and DNA polymerase functions of E. coli DNA polymerase 1 in the translocation of a single-strand break in a DNA strand. Trcuislocation of the breakpoint occurs in the 5 - 3 direction as a result of concomitant nucleotide hydrolysis and polymerization. (B) Template switching involves the extension of a DNA chain at a single-strand break, in a reaction where DNA is duplicated, rather than replaced as in nick treuislation.

A substantial number of observations have demonstrated that the stress-70 proteins bind denatured proteins, as well as some short peptides, and that ATP hydrolysis (possibly with concomitant release of Pj or ADP) results in the release of bound peptides (de Silva et ai, 1990 Hendershot, 1990 Hurtley 1989 Kassenbrock 1988 Palleros etai, 1991). Conversely, bindingof peptides to stress-70 proteins induces ATPase activity above basal levels peptide-induced ATPase activity is often used as a facile in vitro assay for stress-70 protein activity. Schematically, this activity can be subdivided into (1) ATP binding and hydrolysis, followed by product release (P and ADP), (2) peptide binding and release, and (3) a mechanism of coupling peptide binding/release and nucleotide hydrolysis/product release. 

The specific effects of purified dnaj and grpE proteins on the dnaK protein have been characterized (Liberek et al., 1991a). The dnaj and grpE proteins, acting in concert, can stimulate the basal ATPase activity of dnaK 50-fold dnaj appears to enhance the rate of nucleotide hydrolysis by dnaK, but does not substantially affect nucleotide release. The grpE protein appears to enhance the rate of release of nucleotide bound to dnaK. Consequently, neither protein alone exerts a dramatic effect on the overall rate of ATP turnover by dnaK however, the effect of the two proteins combined is to accelerate both the rate of ATP hydrolysis and the rate of nucleotide release, resulting in enhanced ATP turnover. The concentrations of dnaj and grpE required for half-maximal effect are both 0.1-0.2 fiM. 

These mechanisms are not mutually exclusive. Many examples of metallocenter synthesis utilize multiple mechanisms. In addition, it is important to note that metallocenter assembly can be driven by nucleotide hydrolysis reactions, typically involving mechanism 7. L, protein ligand L, covalently modified ligand X, non-metal component of a metallocenter Y, component required for the synthesis of a metal-containing cofactor C, molecular chaperone. 

Kinetic studies indicate that nucleotide hydrolysis precedes electron transfer in the Fe-protein-MoFe-protein complex (12, 13, 114). Although the detailed structural consequences of nucleotide binding on the Fe-protein structure have not been established, it is likely that... 

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