Phosphoenzyme

Acetate kinase is phosphorylated by acetyl phosphate and it has been shown that the phosphoenzyme can synthesise ATP from ADP, and acetyl phosphate from acetate. The mode of decomposition of carbamyl phosphate in aqueous solution is pH dependent and can proceed with either the production of ammonia and carbon dioxide (equation 1), or cyanate (equation 2). No cyanate could be detected during the hydrolysis... 

Mutations in another region, the second cytoplasmic loop between M2 and M3 in Ca-ATPase of sarcoplasmic reticulum (Thr ->Ala, Gly -t Ala, and Glu Gln) also result in a complete loss of Ca-transport and Ca-ATPase activity associated with a dramatic reduction in the rate of phosphoenzyme turnover [96]. These mutations do not affect the affinity of the enzyme for Pj and therefore resemble the Pro mutants [123] in that they affect only the E1P-E2P conformational change and not the affinities for ATP, Ca or Pj. 

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]. 

The reaction starts with the binding of ATP to the H -liganded form of the enzyme, 2H Ei. In the presence of K, this binding is to the K -liganded enzyme form, 2K Ei or 2K E2, or to an occluded form between these two forms. The existence of such an occluded form has not yet been demonstrated, but its detection with filtration or column techniques similar to those used previously to measure occluded transported cations for Na,K-ATPase [113] will be very difficult, because of the rapid dissociation of from the enzyme [96]. Subsequent binding of Mg to 2H E] then leads to phosphorylation at an aspartyl residue [46,114]. The major phosphoenzyme then formed is a K -sensitive intermediate (2H E2-P), whereas a minor part (20%) exists as an ADP-sensitive intermediate (2H Ei-P) [92,93]. With... 

It has been established by substitution of for Mg that, prior to phosphorylation, the divalent cation binds at a cytosolic site with a stoichiometry of about 1 mol per phosphorylation site [124,125]. These experiments also demonstrated that the phosphorylation rate is sensitive to the nature of the divalent cation bound. With Mg bound, the phosphorylation rate is about 20 times faster than with Ca bound. The divalent cation dissociates after dephosphorylation, suggesting that it is tightly bound to the phosphoenzyme during the reaction cycle. It was also demonstrated that the type of divalent cation that occupies the divalent cation site required for phosphorylation is important for the step 2K E2-P to 2K E2 P to 2K E2 [124,125]. With Mg bound, the 2K E2-P conformer is -sensitive, whereas with Ca bound, the intermediate is -insensitive. 

In the absence of K the enzyme exhibits a basal Mg -ATPase activity that can be reduced, but not completely removed, upon further purification of the enzyme by free-flow or zonal electrophoresis [66,89]. Wallmark et al. [104] demonstrated that the rate of spontaneous breakdown of phosphoenzyme corresponded very well to the Mg -ATPase activity at low ATP concentrations, implying that this activity was not due to a contaminating Mg -ATPase with a reaction path independent of the phosphoenzyme. This conclusion was confirmed by Reenstra et al. [129] in a study on the nonhyperbolic ATP dependence of ATPase activity and phosphoenzyme... 

The kinetically deduced existence of two classes of substrate sites may also account for the molar ratio between ATP analogs and inhibitors on the one hand and phosphoenzyme on the other hand. This ratio has been reported to be 2 1 for the ATP analogs adenylyl imido diphosphate (AMP-PNP) [135] and 2, 3 -0-(2,4,6-trinitrophenylcyclohexadienylidine)-ATP (TNP-ATP) [97], and also 2 1 for the ATP-site directed fluorescent inhibitors eosin [99] and FITC [49,50] and the transition-state inhibitor vanadate [126]. 

In the mutants the phosphoenzyme formed from ATP was A DP-sensitive, even under conditions (low concentration and alkaline pH) that caused the accumula-... 

The mutation of ThrlSl, Glyl82, or Glul83 to alanine, or of Glul83 to glutamine also completely inhibited the ATP or acetylphosphate-dependent Ca transport, without effect on the phosphorylation of the enzyme by ATP in the presence of Ca or by Pi in the absence of Ca [127]. The phosphoenzyme formed from ATP retained its ADP-sensitivity at low concentration and alkaline pH, but its rate of decomposition was much slower than that of the wild-type enzyme in the presence of EGTA. These observations implicate the 181-183 region in the conformational changes related to Ca translocation. 

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)... 

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