Enzymes phosphate

Younis, H.M., Weber, G. Boyer, J.S. (1983). Activity and conformational changes in chloroplast coupling factor induced by ion binding Formation of a magnesium-enzyme-phosphate complex. Biochemistry, 22, 2505-12. 

D-Glucose-l-phosphate + enzyme => D-glucose-enzyme + phosphate. 

Bock, A.K. Glasemacher, J. Schmidt, R. Schoenheit, P. Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima. J. BacterioL, 181, 1861-1867 (1999)... 

Reactions 2 and 3, Fig. 4, involving the formation of phosphoryl enzyme through dissociation of binary enzyme-phosphate substrate complexes, are the rate limiting steps in the various reactions catalyzed (40) Inability of such complexes to dissociate when composed of enzyme and PFj-, ADP3-, or ATP4- has been considered in Section III,D,1 and will be further considered in Section III,D,5,c. 

The hydrolysis of fructose 1,6-diphosphate occurs at the oxygen-phosphorus linkage of the substrate, leading to the formation of l80-labeled inorganic phosphate when the hydrolysis is carried out in Ha180. No evidence for an enzyme-phosphate intermediate could be obtained 22). 

The hydrolysis reaction is thought to proceed via a covalent enzyme-phosphate intermediate (112). ... 

Elucidation of the nature of the irreversible enzyme-phosphate linkage evidently would be of the greatest importance for an understanding of the hydrolytic mechanism of ChE s in general. 

Further studies indicate that an ADP-forming acetyl-CoA synthetase is also operative in other extremely thermophilic archaea Pyrococcus woesei, Thermococcus celer, Hyperthermus butylicus, Desulfurococcus amylolyticus), which form acetate as end product of their fermentation [305]. In contrast, in acetate forming (eu)bacteria, acetate formation from acetyl-CoA and the synthesis of ATP from ADP and Pj are catalyzed by two enzymes phosphate acetyltransferase and acetate kinase. This holds true for the extremely thermophilic (eu)bacterium. Thermotoga maritima[3Q5], which ferments... 

The possibility of P-C bond biosynthesis for AEP through a phosphate-phosphonate rearrangement has been tested in Tetrahymena thermophila by growth on a medium containing (D)-[6,6-D2]glucose and isolation of the labelled AEP. The latter, labelled 1,1-D2, was isolated in amounts not consistent with an enzymic phosphate-phosphonate rearrangement of... 

The ATP hydrolytic sites for the proton pump are located at cytosolic sites and the high affinity K+ sites are on luminal face across the membrane [140]. The enzyme is phosphorylated at cytosolic sites by ATP in the presence of Mg2+. Then the enzyme-phosphate complex is dephosphorylated by luminal K+. The kinetic studies carried out by Murakami et al. [139,141] demonstrated that the inhibition of the gastric pump by sofalcone, chalcone and quercetin was competitive with respect to ATP and noncompetitive with K+. In this way, Beil et al. [142] showed that quercetin, flavone and flavanone locked acid formation in parietal cells in response to histamine and cAMP stimulation, flavanone being the most potent inhibitor. H+, K+-ATPase was inhibited by all of them, and this inhibition increased with lowering ATP concentration. The steady-state phosphorylation level of the enzyme was also dose-... 

Inositol monophosphatase catalyses the hydrolysis of a range of phosphate esters of inositol, and in so doing, participates in brain cell chemistry and is believed to be the target for lithium therapy. In the search for inhibitors of the enzyme, phosphate derivatives from 6-0-(2 -hydroxyethyl)cyclohexane-l,2,4,6-tetraol have been prepared, the racemic epoxide (26) acting as a key intermediate. The conversion of (26) into the racemic 1-phosphate (29) via (27) and (28) employed the steps indicated in Scheme 1. Further, the racemic alcohol (27) was... 

The interaction of phosphate with the bovine PAP appears uncomplicated by the intermediacy of the reduced enzyme-phosphate complex, since the red shift and the loss of EPR signal both parallel the loss of catalytic activity (187). Nevertheless, the reduced enzyme-phosphate complex must still be involved because phosphate is a known competitive inhibitor of the bovine enzyme (171). In the bovine case, conversion of the reduced enzyme-phosphate complex to its oxidized counterpart may occur at a rate significantly faster than that observed for the porcine enzyme. The differences between the two enzymes are intriguing and should be examined more closely. 

Evidence for a bridging phosphate in the oxidized enzyme-phosphate complex comes from EXAFS studies on the bovine and porcine enzymes... 

Design an experiment using ATP labeled with in the y position that would suggest that the Na -K+ ATPase reaction involves a stable enzyme-phosphate intermediate. 

If the Na -K ATPase reaction involved a stable enzyme-phosphate intermediate, the mechanism would be a two-step process involving two independent half-reactions ... 

The following experiment would suggest that such an overall reaction occurs. Incubate a fragmented membrane preparation -with K" ion to hydrolyze any phosphate that might be bound to the enzyme. Then wash the membrane preparation to remove all K, and transfer the membrane to a medium containing y-labeled ATP, Na", and Mg ". After a suitable incubation period, wash the membrane preparation to remove any unreacted labeled ATP. Then carry out scintillation counting on the membrane preparation to detect the presence of labeled phosphate. The presence of radioactivity in the membrane fraction would suggest that a stable enzyme-phosphate intermediate had been formed. In fact, a covalent aspartyl phosphate derivative is formed at the active site of the ATPase. 

In the first instance, the inhibitor reacts with the serine hydroxyl moiety of the enzyme to produce the alkylphosphate ester [14]. The formation of this ester is reversible and the enzyme activity may be recovered by nucleophilic attack with compounds such as hydroxylamine [15] and oximes [16]. Alternatively, dealkylation of the enzyme-phosphate may then occur [17], a process known as ageing [18], and the covalent link between the enzyme and the phosphate is stabilised, so preventing any recovery of enzyme activity by oximes [19]. 

FIGURE 11.13 Enzyme-phosphate-metal-substrate complexes. 

They bring about a reversible interconversion of the enzyme-phosphate intermediate between a low energy form, which is at equilibrium with inorganic phosphate, and a high energy form, at equilibrium with ATP. 

Liver Phosphorylase. Liver phosphorylase has been highly purified but not crystallized. It has been shown to contain firmly bound phosphate that does not exchange during activity. The enzyme phosphate is lost on incubation with another inactivating enzyme, and the resulting de-phosphophosphorylase is an inactive protein of about the same size as... 

However, the monofluoro analog 12 has been made the enzyme itself will assemble it fiom Z-fluoro-PEP and S-3-P, as Mark Walker and the Monsanto group have recently shown (Figure 9).3 Actually, EPSP synthase induces formation of 12, it doesn t catalyze it This analog is a powerful inhibitor and the enzyme can t carry it further to fluoro-EPSP thus, turnover and true catalysis do not occur. We have tried to get to this monofluoro derivative fiom the other direction, namely from Z-fluoro-EPSP, but the enzyme will not oblige us. Enzymatic formation of 12 from S-3-P and Z-fluoro-PEP takes less than a minute, but even weeks of incubation of enzyme, phosphate, and Z-fluoro-EPSP give no evidence for formation of 12 or Z-fluoro-PEP.37... 

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