Phosphoenzyme intermediate

The existence of the Ei, and E2 states of the phosphorylated protein, i.e., the high-and low-energy phosphoenzyme intermediate, has been demonstrated by the ATP ADP exchange reaction [92,93] and by the exchange between inorganic phosphate and water [94]. 

Reaction of purified Ca " -ATPase with 0.3 mM NBD-Cl in the presence of 1 mM AMP-PNP and 1 mM CaCl2 caused inhibition of ATPase activity with the incorporation of 2= 15 nmol NBD-Cl per mg protein [335]. The inhibition was attributed to the binding of 7-8 nmol NBD-Cl/mg enzyme protein, corresponding to = 1 mol NBD-Cl per mol ATPase. The NBD-labeled enzyme was digested with pepsin and several NBD-labeled peptides were isolated [335]. All peptides contained the Gly-X (Cys) sequence that occurs only in one place in the Ca -ATPase, i.e., at Gly343-Cys344. Therefore NBD-Cl reacts with the same cysteine 344 residue that is also modified by maleimide derivatives [319]. The NBD modified enzyme had only 5-10% of the ATPase activity of the control ATPase, but the steady state concentration of the phosphoenzyme intermediate was only slightly reduced [335]. The Ca ... 

The rate of phosphoprotein formation in the presence of 5 mM CaCl2 was only slightly affected by mild photooxidation in the presence of Rose Bengal, but the hydrolysis of phosphoenzyme intermediate was inhibited sufficiently to account for the inhibition of ATP hydrolysis [359]. The extent of inhibition was similar whether the turnover of E P was followed after chelation of Ca with EGTA, or after the addition of large excess of unlabeled ATP. These observations point to the participation of functionally important histidine residues in the hydrolysis of phosphoprotein intermediate [359]. 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

Ahn, K. Kornberg, A. Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J. Biol. Chem., 265, 11734-11739 (1990)... 

These P-type ATPases are characterized by phosphoenzyme intermediates, by a conserved consensus sequence, and through inhibition by vanadate ion.537-539 The structures are poorly known. Some consist of single chains (perhaps dimerized) and some have more than one chain. However, the major subunit always appears to have about ten transmembrane helices with a large 430-residue cytoplasmic domain between the fourth and fifth helices. This domain contains the ATP binding site and the phosphoaspartyl group of the phosphoenzyme.534 This is Asp 369 for the Na+,K+- ATPase. 

Most kinases transfer chiral phospho groups with inversion and fail to catalyze partial exchange reactions that would indicate phosphoenzyme intermediates. However, nucleoside diphosphate kinase contains an active site histidine which is phosphorylated to form a phosphoenzyme.869 The enzyme catalyzes phosphorylation of nucleoside diphosphates other than ADP by a nucleotide triphosphate, usually ATP. 

There is evidence (but not conclusive evidence) that the mechanisms involves direct phosphoryl transfer to water rather than the formation of a phosphoenzyme intermediate.293 This utilizes the fact that ATP also serves as a substrate to inorganic pyrophosphatase. Hydrolysis of the ATP analogue adenosine 5 -0-(3-thiotriphosphate) chirally labelled with I70 and I80 at the y-phosphate proceeds with inversion of configuration to give the chiral [170,180]-thiophosphate (Figure 13).293... 

Phosphoglucomutase catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate via a phosphoenzyme intermediate in which the phosphate is bound to a serine residue at the active site. The Mg2+ is not bound to the phosphate, but studies with the Nin-substituted enzyme suggest the presence of a second-sphere complex between phosphate and metal, which could ensure correct positioning of the phosphoryl group for nucleophilic attack, as shown in Figure 14.278... 

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...

Trinitrophenyl-nucleotides constitute a unique class of fluorescent ATP-ana-logs, since they bind at the catalytic site of the phosphoenzy me of the Ca2+-ATPase after departure of ADP and give off a tremendous fluorescence signal upon conversion of the phosphoenzyme intermediate from ADP-sensitive to ADP-insen-sitive (c.f., Figure 6), possibly reflecting a hydrophilic-hydrophobic transition related to closure of the catalytic site (Andersen et al., 1985 Seebregts and McIntosh, 1989). 

Figure 22. Analysis of the rates of dephosphorylation of phosphoenzyme intermediates of wild-type and Pro312 mutants of the SR Ca2+-ATPase. Left panel Phosphorylation was performed with [y-32P]ATP in the presence of Ca2+. The buffer conditions were adjusted to obtain predominantly E,P in steady state as indicated by the complete disappearance of the phosphoenzyme upon addition of ADP. EGTA was added to terminate phosphorylation by chelation of Ca2+ and permit observation of the dephosphorylation of E,P through conversion to E2Pand hydrolysis of the latter intermediate. A rapid dephosphorylation is observed with the wild type, while in the mutants the dephosphorylation of E,P is inhibited. More than 80% of the phosphoenzyme remains five minutes after addition of EGTA in the Pro312- Ala mutant. Right panel Phosphorylation was performed with [32P]Pj by the backdoor reaction in the absence of Ca2+...

Na+, K+-ATPase was inactivated by FSB A in the presence of Mg23-. Although Na+ and K+ are not essential for inactivation, they accelerate the inactivation rate. In the absence of Mg2+, the enzyme was not inactivated by FSB A even in the presence of both Na+ and K+. Sequence study on the FSBA-modified dog kidney enzyme revealed that one of the major labeled sites is a lysyl residue equivalent to Lys-725 of the torpedo enzyme. The simultaneous replacement of both Lys-712 and Lys-713 by Met of SR-ATPase (The two lysyl residues are equivalent to Lys-725 and Lys-726 of the torpedo Na+, K+-ATPase) did not change the Ca2+-transporting activity or the formation of phosphoenzyme intermediate,781 indicating that these lysyl residues are not essential for enzyme activity. 

A phosphoenzyme intermediate is formed in one type of covalent catalysis in enzymes ... 

The mechanism of the PTP hydrolysis reaction has two steps. First, phosphate is transferred from tyrosine to the cysteine residue of the P-loop, which generates a phosphoenzyme intermediate with concomitant release of tyrosine. This process is followed by hydrolysis of the phosphoenzyme to free enzyme and inorganic phosphate. Two active site residues are of primary importance during the catalytic cycle the nucleophilic cysteine of the P-loop and an aspartate on a nearby flexible loop, which serves as a general acid/base catalyst (Fig. 3). After attack of the cysteine on phosphotyrosine, tyrosine can be expelled as the protonated phenol after proton donation by the catalytic aspartic acid, which forms the phosphoenzyme intermediate and free tyrosine. The aspartate anion then deprotonates the hydrolytic water molecule that attacks phosphocysteine, which liberates inorganic phosphate (Fig. 4) (9). 

Resolution of the phosphoenzyme intermediate into free enzyme and inorganic phosphate is accomplished by orientation and deprotonation of water by the aspartate residue and attack at the phosphothioester. Mutagenesis of the aspartate residue to alanine (as well as the Arg in the P-loop) shows its importance in this second step, as it leads to accumulation of the phosphoenzyme intermediate (12). 

Protein phosphatases that are specific for phosphoserine/ phosphothreonine have a distinct reaction mechanism from tyrosine phosphatases. Protein serine phosphatases are transition metal-dependent, and the reaction mechanism does not involve a phosphoenzyme intermediate as in the case of PTPs. Crystal structures of multiple protein serine phosphatases have revealed how the enzymes catalyze hydrolysis of phosphoserine (14). 

Stabilization of the phosphoenzyme intermediate that is somewhere in between the other two cases. 

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