Phosphorylation partial reactions

Partial reactions not dependent on photosystem II, such as cyclic phosphorylation or the photoreduction of NADP with an electron donor that circumvents photosystem II (ascorbate + DPIP), are either not inhibited or inhibited only weakly. These herbicides also do not inhibit mitochondrial oxidative phosphorylation. 

CPSase catalyzes the formation of carbamyl phosphate from glutamine, bicarbonate, and two equivalents of ATP. The biosynthesis involves four partial reactions. GLNase catalyzes the formation of ammonia from glutamine. The remaining three partial reactions are catalyzed by SYNase. Bicarbonate is activated by ATP to form carboxyphosphate, which reacts with ammonia to form carbamate. The ATP-dependent phosphorylation of carbamate results in the production of carbamyl phosphate. 

Our present understanding of the phosphorylation reaction has been greatly aided by the ability to study several partial reactions of the overall process, and follow them during fractionation of the intact thylakoid vesicle. 

Examination of the first partial reaction reveals that the mutase functions as a phosphatase—it converts 2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the phosphoryl group remains linked to the enzyme. This phosphoryl group is then transferred to 3-phosphoglycerate to reform 2,3-bisphosphoglycerate. 

ATPase activity and exchange reactions, such as P,-ATP exchange, are partial reactions of oxidative and photosynthetic phosphorylation. These reactions have been described in detail and have been considered to consist of a series of reversible chemical reactions forming high energy intermediates (X-Y and X-P) [104-106]. In... 

An effort is underway in our laboratory to determine transition state structures for phosphoryl transfer reactions by the use of secondary 0 isotope effects measured by the remote label method. The hypothesis is that an associative mechanism would have single bonds from phosphorus to the nonbridge oxygens in the transition state, whereas a dissociative mechanism would have enhanced bond order. So far we have determined the secondary 0 isotope effects on glucose-6-P hydrolysis at pH 4.5, 100°C (112), and by alkaline phosphatase (128). The chemical hydrolysis showed no isotope effect on P-O bond cleavage, which is consistent with a largely dissociative transition state without total conservation of bond order to phosphorus (i.e., a partial positive change exists on phosphorus). 

Partial reaction of phosphoryl chloride with amines can be used for synthesis of phosphonic acids 392,393 if applied to phosphonic dichlorides it affords unsymmetrical phosphinic acids.396... 

The detection and characterization of intermediates in complex reactions is often accomplished through the use of isotope-exchange studies. In the case of enzymic phosphoryl transfer reactions, the presence of a phosphorylated enzyme intermediate is implicated by a partial exchange process as exemplified by the following reaction of hexokinase (Knowles, 1980) ... 

Another consequence of partial lipid depletion is that the residual K -stimulated phosphatase is not inhibited by Na nor undergoes stimulation by Na" " -I-ATP at low K concentrations [117,124]. This has led to the suggestion that the K" -stimulated phosphatase activity in preparations containing a full lipid complement is a monomeric enzyme function by virtue of a K -induced dissociation of the dimer into monomers. Na would inhibit the phosphatase by inducing dimerization. In lipid-depleted preparations, having only one of the monomers saturated with lipid as a prerequisite for phosphatase activity to occur, Na would be unable to cause dimerization. Hence, it does neither inhibit the phosphatase activity nor stimulate Na-K ATPase activity for which dimerization is essential. As a consequence, the Na -dependent reactions (ATP-phosphorylation and the ADP-ATP exchange) would be expressions of the dimer and the -dependent reactions (K -stimulated dephosphorylation and phosphatase) expressions of the monomer. However, thimerosal, which would inhibit monomer interaction, does not inhibit any of these partial reactions, but increases the affinities for K" " [125,126] and lipid depletion reduces the Na -dependent phosphorylation less than the overall Na-K ATPase activity [122]. It is clear from these studies that an unequivocal answer as to the monomeric and dimeric nature of the partial reactions in the overall Na-K ATPase reaction and their lipid dependence must await determination of their functional molecular weights. 

Some studies have been carried out on the role of phosphorylated intermediates and the role of partial reactions, subunit structure and involvement of phospholipids. All these studies have been done with crude preparations from gastric mucosa, which means that the preparation contains (K -I- H )-ATPase activity (see Section 3). This makes it difficult to relate these findings to the anion-sensitive ATPase. 

The hypothesis states that the proximity to the GLN domain determines the reaction catalyzed by the CPS subdomain. The GLN domain is in intimate contact with CPS.A but there are no direct interactions with CPS.B. According to this interpretation, CPS.A catalyzes the bicarbonate dependent ATPase reaction because of this proximity to the source of ammonia, while CPS.B, which is far from the GLN domain, catalyzes the phosphorylation of carbamate. To test the idea that the GLN domain directs the function of the CPS domain, the mutagenesis experiments previously conducted with E. coli CPSase (55) were repeated with the same results. However, if the GLN domain is removed and a disabling mutation is introduced into CPS.A, rather than selectively abolishing the ATPase activity, both the ATPase and ATP synthetase reactions were inhibited by approximately 50%. These results suggest that, in the absence of the GLN domain, CPS.A and CPS.B of the intact . coli CPSase can catalyze both ATP dependent partial reactions. However, when the CPS subunit is associated with the GLN domain, CPS.A is specialized for the activation of bicarbonate while CPS.B phosphorylates carbamate. Thus, we have the interesting situation in which the role of two functionally equivalent domains is determined by their juxtaposition relative to a third domain in the complex. 

Once the nature of the phosphoamino add is established, its catalytic competence has to be demonstrated. This can be done by kinetic experiments demonstrating the existence of double-displacement (or ping-pong) kinetics. However, if sequential kinetics is observ it will have to be own that the rates of the partial reactions leading to phosphorylation or dephosphorylation are comparable to the overall catalytic rate (Bridget et al., 1968 Wimmer and Rose, 1978). Not in all cases where a phosphoamino acid was identified could this condition be fulfilled, as is demonstrated clearly by penetrating studies on hexokinase (Wimmer and Rose, 1978). 

Chemical modification studies with fluorescein-5 -isothiocyanate support the proximity of Lys515 to the ATP binding site [98,113-117,212,339]. Fluorescein-5 -isothiocyanate stoichiometrically reacts with the Ca -ATPase in intact or solubilized sarcoplasmic reticulum at a mildly alkaline pH, causing inhibition of ATPase activity, ATP-dependent Ca transport, and the phosphorylation of the Ca " -ATPase by ATP the Ca uptake energized by acetylphosphate, carbamylphos-phate or j -nitrophenyl phosphate is only partially inhibited [113,114,212,339]. The reaction of -ATPase with FITC is competitively inhibited by ATP, AMPPNP, TNP-ATP, and less effectively by ADP or ITP the concentrations of the various nucleotides required for protection are consistent with their affinities for the ATP binding site of the Ca -ATPase [114,212,340]. 

No details are given for scheme A. Presumably one could use the phosphoryl chloride instead of the fluoride. Scheme B, in which ethyl chloride is formed, was run in boiling xylene using equimolar quantities of the reactants. Michaelis (33) has partially described the preparation of starting materials from secondary amines with phosphorus oxychloride and also ethyl dichlorophosphate. Schrader (38) obtained alkyl and amido fluophosphates by reaction of the corresponding chlorophosphates with sodium fluoride in aqueous or alcoholic solution. 

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