Orthophosphate, transfer

In contrast to tyrosine kinases, Tyrosine phosphatases (PTPs) are enzymes which act on phosphorylated proteins and catalyze the transfer of a phosphate group from a tyrosine residue to a water molecule, generating orthophosphates in a process which is referred to as dephosphorylation. PTPs are involved in many cellular signal transduction pathways. 

L-Ornithine transcarbamoylase catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 29-9). While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citmlline take place in the cytosol. Entry of ornithine into mitochondria... 

The hydrated sulfate S04(H20)2 could be produced by electrospray in the gas phase,80 but neither the triply charged orthophosphate P04 nor the doubly charged HOPOj" were observed as the naked ion or the hydrate.81 CID of the hydrated sulfate led to simple desolvation down to n = r = 4. The decomposition of the r = 4 hydrate led to charge reduction by intracluster proton transfer ... 

In the malachite green procedure, 10 ml of the sample solution containing up to 0.7 xg phosphorus as orthophosphate was transferred into a 25 ml test tube. To this solution was added 1 ml each of 4.5 M sulfuric acid and the reagent solution. The solution was shaken with 5 ml of a 1 3 v/v mixture of toluene and 4-methylpentan-2-one for 5 min. After phase separation, the absorbance of the organic phase was measured at 630 nm against a reagent blank in 1 cm cells. 

Although chromate is the best aqueous corrosion inhibitor available, its use has been severely curtailed due to toxicity and environmental concerns ( ). One of the more successful non-chromate treatments involves the use of phosphate/phosphonate combinations. This treatment employs high levels of orthophosphate to promote passivation of the metal surfaces. Therefore, it is important to control calcium phosphate crystallization so that high levels of orthophosphate may be maintained in the system without fouling or impeding heat-transfer functions. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

In both schemes, the specificities of the pump for catalysis change in the two enzyme states. Jencks points out that coupling is determined (a) by the chemical specificity achieved in catalyzing phosphoryl transfer to and from the enzyme (wherein E-Ca2 reversibly binds ATP, and E reacts reversibly with orthophosphate), and (b) by the vectorial specificity for ion binding and dissociation (wherein E reversibly binds/dissociates cytoplasmic calcium ion, and E—P reversibly binds/dissociates luminal calcium). There must be a single conformation change during the reaction cycle between Ei and E2 in the free enzyme and from Ei P-Ca2 to E2-P-Ca2 after enzyme phosphorylation. 

Phosphorus stock standard solution, 1000 pg P O mh (436 pg P mM) -dissolve 1.9173 g potassium dihydrogen orthophosphate, previously dried for 1 h at 105°C, in water. Transfer with beaker washings to a 1-1 volumetric flask, make up to the mark and mix. 

Potassium stock standard solution, 1000 pg K mb - dry potassium dihydrogen orthophosphate (KH2PO4) for 2 h at 105°C, then allow to cool in a desiccator. Weigh 2.889 g into a 100-ml beaker, add sufficient water to dissolve, and transfer to a 1-1 volumetric flask with beaker washings make up to the mark with water and mix. 

Calcium phosphate will ordinarily precipitate at concentrations typically exceeding 5 ppm PO4 or less, forming amorphous calcium orthophosphate (tricalcium phosphate) sludge, Ca3(P04>2, in the bulk water and crystalline hydroxyapatite, Caio(OH)2(P04)2, at heat-transfer surfaces. 

Let us consider the mechanism of glyceraldehyde 3-phosphate dehydrogenase in detail (Figure 16.8). In step 1, the aldehyde substrate reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal. Step 2 is the transfer of a hydride ion to a molecule of NAD + that is tightly bound to the enzyme and is adjacent to the cysteine residue. This reaction is favored by the deprotonation of the hemithioacetal by histidine 176. The products of this reaction are the reduced coenzyme NADH and a thioester intermediate. This thioester intermediate has a free energy close to that of the reactants. In step 3, orthophosphate attacks the thioester to form 1,3-BPG and free the cysteine residue. This displacement occurs only after the NADH formed from the aldehyde oxidation has left the enzyme and been replaced by a second NAD+. The positive charge on the NAD+ may help polarize the thioester intermediate to facilitate the attack by orthophosphate. 

Pyrophosphate is then hydrolyzed to orthophosphate, and so the equivalents of two compounds having high phosphoryl transfer potential are consumed in the activation of acetate. We will return to this type of activation reaction in fatty acid degradation (Section 22.2.2). where it is used to form fatty acyl CoA, and in protein synthesis, where it is used to link amino acids to transfer RNAs (Section 29.2.1). 

Figure 21.8. Phosphorylase Mechanism. A bound HP042- group (red) favors the cleavage of the glycosidic bond by donating a proton to the departing glucose (black). This reaction results in the formation of a carbocation and is favored by the transfer of a proton from the protonated phosphate group of the bound pyridoxal phosphate PLP group (blue). The combination of the carbocation and the orthophosphate results in the formation of glucose 1-phosphate.

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