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Phosphorylated intermediate

A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. [Pg.13]

The T2 site also became protected from tryptic hydrolysis after phosphorylation of the native or solubilized sarcoplasmic reticulum vesicles with inorganic phosphate in a calcium free medium in the presence of dimethylsulfoxide or glycerol [121,252]. Under these conditions the Ca -ATPase is converted into a covalent E2-P intermediate, that is analogous in conformation to the E2V intermediate formed in the presence of vanadate. In contrast to this, the T2 site in the stable phosphorylated Ca2E P intermediate generated by the reaction of the Ca -ATPase with chro-mium-ATP in the presence of Ca [178,253] was fully exposed to trypsin, just as it was in the nonphosphorylated Ca2Ei form. Therefore the phosphorylated intermediates show the same sensitivity to trypsin at the T2 site as the corresponding nonphosphorylated enzyme forms. [Pg.86]

The fact that the 3,P-NMR signal of 183a can only be observed in pyridine-containing solution provides food for thought124). Viewed in conjugation with the idea that alkyl metaphosphates could form adducts such as 173 and 174 U9,120) as discussed above, formulation as a zwitterionic pyridine/metaphosphate adduct (188) seems reasonable. Similar adducts have also been found in the reaction of TPS with dinucleotides and trinucleoside diphosphate 126). In any case, the reactions of 183 or 188 are in full accord with the expected properties of a monomeric metaphosphate and its reactivity towards alcohols is far greater than that of all other reactive phosphorylation intermediates which can arise on reaction of TPS with oligonucleotides 126). [Pg.116]

Mahaney, J. E., Thomas, D. D. and Froehlich, J. P. The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model. Biochemistry 43 4400-4416, 2004. [Pg.92]

The clearest example of rapid turnover of a functional micelle is a reaction of a micellized gem-diol. Attack of the anion of the gem-diol gives an acetylated or phosphorylated intermediate which rapidly breaks down regenerating the catalytic gem-diol (Scheme 8) (Menger and Whitesell, 1985). [Pg.260]

Nevertheless, using GC-based technologies, the quantification of several important intermediates of central metabolism, especially phosphorylated intermediates, is not very reliable, presumably because these compounds and their derivatives are not thermostable. For an analysis of these groups of metabolites, an LC-MS (liquid chromatography or HPLC coupled to MS) is more suitable, because it eliminates the need for volatility and thermostability and thereby eliminates the need for derivatization. Using a triple quadrupole MS, most of the intermediates in glycolysis, in the pentose phosphate pathway, and in the tricarboxylic acid cycle were measured in E. coli [214]. [Pg.150]

In contrast to experiments which attempted to identify phosphorylated intermediates on the look and see what is there basis, others were designed to investigate possible pathways of glucose breakdown suggested from its known chemical reactivity. Treating glucose in vitro... [Pg.51]

Earlier work by Nancy Bucher showed an ATP requirement for cholesterol biosynthesis. The involvement of phosphorylated intermediates was established by Comforth, Popjak, and their associates in the early 1960s with the discovery of kinases which successively phosphorylated MVA to MVA-P and MVA-P to MVA-PP. MVA-PP was decar-boxylated and dehydrated to give the biological C5 isoprene unit, isopentenyl pyrophosphate. This undergoes successive head-to-tail condensations to give the linear 15C terpenoid, famesyl pyrophosphate. [Pg.135]

In the interaction of isoflurophate with AChE, a phosphorylated intermediate is formed and fluoride is released. An important characteristic of the organophos-phate-induced inhibition is that the bond between the phosphate and the enzyme is very stable. While the regeneration of most carbamylated enzymes occurs with a half-life of minutes or hours, the recovery of a phosphorylated enzyme is generally measured in days. These agents are referred to, therefore, as irreversible inhibitors. [Pg.127]

The two-step mechanism of phosphate ester hydrolysis by the (Znn)2-containing alkaline phosphatase (AP) (7) is thus somewhat mimicked by 24. The phosphoryl intermediate 25 is generated by nucleophilic attack of the alkoxide moiety in 24b at BNP" and is hydrolyzed by the intramolecular Zn11—OH" species in 25b. Thus, the attack at the BNP... [Pg.242]

Colomb, M.G. Cheruy, A. Vignais, P.V. Nucleoside diphosphokinase from beef heart cytosol. II. Characterization of the phosphorylated intermediate. Biochemistry, 11, 3378-3386 (1972)... [Pg.535]

In contrast to the hydrolysis and synthesis of ATP connected with proton translocation in mitochondria, chloroplasts and bacterial membranes, the energy linked movement of calcium ions gives rise to the appearance of an acid-stable phosphorylated intermediate in the membranes. A cation specific phosphorylation also occurs in the membranes of the sodium potassium transport system183. However, due to the inability to correlate phosphorylation and ion movement in the latter membranes, membrane phosphorylation has been questioned as being a step in the reaction sequence of ion translocation184,18s. Solely the sarcoplasmic calcium transport system allows to correlate directly and quantitatively ion translocation with the phosphoryl transfer reactions. [Pg.40]

Due to the formation of a phosphorylated intermediate with a lower calcium affinity, calcium is released from the protein. It remains to be shown that this transient affinity change is comparable to the affinity difference which is required to explain the concentration of calcium ions by more than a factor of 1000 as it occurs during active calcium accumulation. [Pg.41]

The initial step in the sequence which leads to ATP synthesis during calcium release is the incorporation of inorganic phosphate into the transport protein. It was first demonstrated in experiments with sarcoplasmic reticulum vesicles which were actively loaded with calcium phosphate193,194). The membranes of the calcium loaded vesicles rapidly incorporate inorganic phosphate when the concentration of ionized calcium in the assay is reduced by the addition of EGTA. The involvement of this phosphorylated intermediate in ATP synthesis infers from the finding that on addition of ADP the level of phosphoenzyme drops and simultaneously, calcium is released and ATP is synthesized193,194 (Fig. 15). The same observations have been made when the... [Pg.43]

Table 11. Properties of the phosphorylated intermediates formed when inorganic phosphate is incorporated into the transport protein... Table 11. Properties of the phosphorylated intermediates formed when inorganic phosphate is incorporated into the transport protein...
Since the properties of the two phosphorylated intermediates make it difficult to find experimental conditions under which both species exist simultaneously in suf-... [Pg.50]

Importance of Phosphorylated Intermediates Each of the nine glycolytic intermediates between glucose and pyruvate is phosphorylated (Fig. 14-2). The phosphoryl groups appear to have three functions. [Pg.525]

Because the plasma membrane generally lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, despite the large difference in their intracellular and extracellular concentrations. [Pg.525]

The biological roles of phosphorus include (1) anabolic and catabolic reactions, as exemplified by its essentiality in high-energy bond formation, e.g., ATP (adenosine triphosphate), ADP (adenosine diphosphate), etc., and the formation of phosphorylated intermediates in carbohydrate metabolism ... [Pg.1282]

The rapid oxygen exchange is consistent with a mechanism involving a phosphorylated intermediate. Numerous attempts have been made to directly detect and isolate such an intermediate using high specific... [Pg.538]

Ribonuclease A hydrolyzes RNA adjacent to pyrimidine bases. The reaction proceeds through a 2, 3 -phosphate cyclic diester intermediate. Formation and breakdown of the cyclic diester appear to be promoted by concerted general-base and general-acid catalysis by two histidine residues, and by electrostatic interactions with two lysines. These reactions proceed through pentavalent phosphoryl intermediates. The geometry of these intermediates resembles the geometry of vanadate compounds that act as inhibitors of the enzyme. [Pg.172]

Processes of this type have been realized in supramolecular phosphorylation reactions. Indeed, the same [24]-N6C>2 macrocycle 38 as that already used in the studies of ATP hydrolysis was also found [5.60] to mediate the synthesis of pyrophosphate from acetylphosphate (AcP). Substrate consumption was accelerated and catalytic with turnover following the steps (1) substrate AcP binding by the proto-nated molecular catalyst 38 (2) phosphorylation of 38 within the supramolecular complex, giving the phosphorylated intermediate PN 81 (3) binding of the substrate HP042 (P) (4) phosphoryl transfer from PN to P with formation of pyrophosphate PP (Fig. 8) (5) release of the product and of the free catalyst for a new cycle [5.60]. PP is also formed in the hydrolysis of ATP in the presence of divalent metal ions [5.61]. [Pg.62]

Fig. 8. Cocatalysis pyrophosphate synthesis by phosphoryl transfer mediated by macrocycle 38 via the phosphorylated intermediate 81 [5.12]. Fig. 8. Cocatalysis pyrophosphate synthesis by phosphoryl transfer mediated by macrocycle 38 via the phosphorylated intermediate 81 [5.12].
The ATPase has an absolute requirement for Mg2+ which is essential for the breakdown of the phosphorylated intermediate. There is evidence for at least three forms of the phosphoenzyme, only the last of which is sensitive to Mg2+.144 Monovalent cations are effective as activators in the sequence K+ Tl+ > Na+ > NH4+ > Rb+ Cs4 > Li1, again through the promotion of the loss of phosphate from the phosphorylated intermediate.145 It has been postulated that K+ may be involved in antiport with Ca2+ to maintain charge balance, but it is currently thought that this involves protons or a flux of anions. [Pg.566]

Acid and alkaline phosphatases with phosphorylated intermediates are inhibited by vanadate. This has been exploited in the study of the role of alkaline phosphatase in mineralization.1069 Vanadate also inhibits the ATP-dependent degradation of proteins in reticulocytes.1070... [Pg.665]

Figure 17. Analysis of phosphoenzyme intermediates of SR Ca2+-ATPase mutants with alterations to carboxylate-containing residues in the transmembrane sector. Wild-type or mutant Ca2+-ATPases expressed in the endoplasmic reticulum membranes of COS-1 cells were phosphorylated with [y-32P] ATP (panel a) or [32P]P (panels b and c). Following acid-quench of the phosphorylated intermediate, the samples were subjected to SDS-polyacrylamide gel electrophoresis under acid pH conditions and the dried gels were autoradiographed to visualize the radioactivity associated with the covalently bound phosphate. Panel a shows the Ca2+-concentration dependence of phosphorylation from ATP. The Glu309- Lys mutant is unable to phosphorylate, even at 12.5 mM Ca2+. In the wild-type Ca2+-ATPase the phosphorylation reaction is fully saturated at 10 pM Ca2+. Panel b shows lack of Ca2+ inhibition of backdoor phosphorylation from P in the mutants. E indicates the presence of ECTA to chelate Ca2+ (normally a requirement for phosphorylation by the backdoor route). C indicates the... Figure 17. Analysis of phosphoenzyme intermediates of SR Ca2+-ATPase mutants with alterations to carboxylate-containing residues in the transmembrane sector. Wild-type or mutant Ca2+-ATPases expressed in the endoplasmic reticulum membranes of COS-1 cells were phosphorylated with [y-32P] ATP (panel a) or [32P]P (panels b and c). Following acid-quench of the phosphorylated intermediate, the samples were subjected to SDS-polyacrylamide gel electrophoresis under acid pH conditions and the dried gels were autoradiographed to visualize the radioactivity associated with the covalently bound phosphate. Panel a shows the Ca2+-concentration dependence of phosphorylation from ATP. The Glu309- Lys mutant is unable to phosphorylate, even at 12.5 mM Ca2+. In the wild-type Ca2+-ATPase the phosphorylation reaction is fully saturated at 10 pM Ca2+. Panel b shows lack of Ca2+ inhibition of backdoor phosphorylation from P in the mutants. E indicates the presence of ECTA to chelate Ca2+ (normally a requirement for phosphorylation by the backdoor route). C indicates the...

See other pages where Phosphorylated intermediate is mentioned: [Pg.130]    [Pg.1508]    [Pg.298]    [Pg.92]    [Pg.72]    [Pg.127]    [Pg.346]    [Pg.40]    [Pg.41]    [Pg.41]    [Pg.44]    [Pg.104]    [Pg.477]    [Pg.968]    [Pg.361]    [Pg.303]    [Pg.168]    [Pg.406]    [Pg.268]    [Pg.268]    [Pg.341]    [Pg.188]    [Pg.433]    [Pg.20]   


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