GTP hydrolyzing enzymes

Stimulation and inhibition of the enzyme by the GPCR-G-protein cycle occur by analogous mechanisms. Agonists induce hormone receptors to increase a Ga-GDP-GTP exchange and subsequent Ga 3y dissociation (GDP-a py + GTP GTP-ax + [3y + GDP) (Fig. 4). Consequently, agents that affect either the dissociation of either G or Gs, or the association of their respective as, a , or (3y subunits with adenylyl cyclase could affect rates of cAMP formation in enzyme preparations or in intact cells and tissues. There are several important examples. Gas is stably activated by poorly hydrolyzable analogs of GTP, e.g. GTPyS... 

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]. 

This reaction then switches on the effector molecule, and the signal is relayed along the pathway (see Fig. 2.7). When the enzyme GTPase hydrolyzes GTP to GDP and removes the phosphate group, the trimeric subunits change back to the inactivated state. This receptor is once again ready to receive and transmit further signals. 

Biain adenosinetriphosphatase This enzymatic activity is persistendy associated with brain micFotubules even after multiple cycles of warm-induced microtubule assembly, centrifugation to separate protomer and polymer, cold-induced disassembly, and subsequent centrifugation to remove cold-stable aggregates (White et aL, 1980). The enzyme hydrolyzes boA GTP and ATP, and recent work by Tominaga and Kaziro (1982) indicates that there are two distinct ATP-ases, one that is of low M, (around 33,000) and tubulin dependent in the presence of calcium ion, and the other of larger size and associated with membrane vesicles... 

This enzyme [EC 3.5.4.25] catalyzes the reaction of GTP with three water molecules to produce formate, 2,5-dia-mino-6-hydroxy-4-(5-phosphoribosylamino)pyrrmidine, and pyrophosphate (or, diphosphate). In this reaction, two C-N bonds are hydrolyzed, releasing formate, with... 

Nonhydrolyzable GTP Analogs Many enzymes can hydrolyze GTP between the j3 and y phosphates. The GTP analog j3,y-imidoguanosine 5 -triphosphate Gpp(NH)p, shown below, cannot be hydrolyzed between the j3 and y phosphates. Predict the effect of microinjection of Gpp(NH)p into a myocyte on the cell s response to /3-adrenergic stimulation. 

The enzyme has been partially purified (70-fold) from 38,000 X 9 supernatant fluid from sheep brain homogenates by Ipata (55-58). Thq enzyme (MW 140,000) is reported to be specific for 5 -AMP and 5 -IMP although the substrate specificity does not appear to have been examined closely. 2 - and 3 -AMP are not hydrolyzed (56). Unlike the enzyme from many sources the brain enzyme does not require divalent cations and indeed Co2+, which stimulates several other 5 -nucleotidases, was inhibitory at 5 mM. The enzyme is strongly inhibited by very low concentrations of ATP, UTP, and CTP (50% inhibition by 0.3 pM ATP) but not by GTP. 2 -AMP, 3 -AMP, and a variety of other nucleoside monophosphates, nucleosides, and sugar phosphates do not inhibit. A kinetic examination of ATP, UTP, and CTP inhibition (56-58) revealed that inhibition curves were sigmoidal, indicating cooperativity between inhibitor molecules and an allosteric type of interaction between inhibitor and protein. The metabolic significance of ATP inhibition is... 

As discussed briefly in Section I,A, glucose-6-phosphatase is now known to be a multifunctional enzyme capable of catalyzing potent phosphotransferase as well as phosphohydrolase reactions [see Eqs. (1)—(4) ]. Compounds demonstrated to function as effective phosphoryl donors include fructose-6-P (30), mannose-6-P (40), PPi (35-38), a variety of nucleosidetriphosphates and nucleosidediphosphates—most effectively CTP, CDP, deoxy-CTP, ATP, ADP, GTP, GDP, and ITP (41, 45)— carbamyl-P (43), phosphoramidate (44), phosphopyruvate (42, 43) and glucose-6-P itself (30, 31). The various phosphoryl donors are also hydrolyzed by action of the enzyme (see preceding references). Eqqa-tions (1)—(4), which describe these various activities, are given in Section I,A. 

Answer A hormone can be degraded by extracellular enzymes (such as acetylcholinesterase). The GTP bound to a G protein can be hydrolyzed to GDP. A second messenger can be degraded (cAMP, cGMP), further metabolized (IP3), or resequestered (Ca2 +, in the endoplasmic reticulum). A receptor can be desensitized (acetylcholine receptor/channel), phosphorylated/inactivated, bound to an arrestin, or removed from the surface ( -adrenergic receptor, rhodopsin). 

M thermoautotrophicum cells synthesize ATP in the presence of an artificially imposed pH gradient [18], Proton uptake is not detected following acidification. The addition of valinomycin results in the synthesis of ATP and is accompanied by the extrusion of K but not protons. ATP synthesis is unaffected by DCCD and is stimulated by uncouplers such as 2,4-dinitrophenol and m-chlorophenyl hydrazone. Membrane vesicles from M thermoautotrophicum synthesize ATP when conditions are anaerobic in response to the membrane potential since the addition of suppresses synthesis. ATP synthesis is inhibited by 100 pM CCCP and partially inhibited by DCCD (53% at 100 pM). ATP synthesis also takes place in response to a ApH produced by the oxidation of hydrogen. In this case, ATP synthesis is inhibited by lOpM DCCD and CCCP. Unlike cells, vesicles do not synthesize ATP in response to an artificially imposed ApH or in the presence of valinomycin [54]. M. thermoautotrophicum membranes have an ATPase activity that hydrolyzes ATP, GTP, and UTP at approximately the same rate. The enzyme loses activity at -90°C which is due to aggregation, and activity is restored following sonication. ATPase activity is partially inhibited by DCCD (40% at lOOpM) when membranes are incubated at pH 8 for 10 min [18]. A similar ATPase is found in a different strain of M. thermoautotrophicum [29]. The enzyme is most active at an alkaline pH and it is not significantly inhibited by ADP, 5mM NEM, or 150 pM DCCD. The absence of NEM inhibition suggests that the enzyme may not be a V-type ATPase. 

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