Phosphotransferases

The stereochemical consequence of [lsO]thiophosphoryl and [l60, nO, lsO]phos-phoryl group transfer catalyzed by seven phosphotransferases were simultaneously determined in the author s laboratory and in the laboratory of J.R. Knowles. The first to be completed was the demonstration of inversion by adenylate kinase however, prior to that glycerokinase, hexokinase and pyruvate kinase had been shown to catalyze [l80]thiophosphoryl group transfer with the same stereochemical consequences, either all with inversion or all with retention. Glycerokinase was later shown to catalyze both [l60, nO, l80]phosphoryl and [l80]thiophosphoryl group 

The stereochemical courses of the glycerokinase, pyruvate kinase, and hexokinase reactions were elucidated simultaneously in a two-stage study. The enzymes first 

In the second stage of the study glycerokinase was shown to catalyze both phosphoryl and thiophosphoryl transfer with inversion of configuration. In the 

Methods similar to those employed for glycerokinase utilizing chiral 

Phosphorylated compounds, e.g., the esters, amides, and anhydrides of phosphoric acid, participate in many secondary reactions. High-energy and low-energy phosphates may be distinguished. The former liberate up to 13,000 cal/mol if the bond between the phosphate residue and the acceptor molecule is hydrolyzed, the latter set free about 3,000 cal/mol (Table 13). 

The energy content of the phosphate bond depends to a considerable extent on how much the electron resonance in the phosphorylated compound is disturbed. In phosphates with low-energy content the resonance of the phosphate anion, which depends on the mutual convertibility of the mesomeric structures I-IV  

In the case of energy-rich phosphates electron resonance is also hindered in the acceptor molecule. For example 

The decrease of resonance causes bond polarization. The stronger this polarization, the easier electrophilic or nucleophilic substitution of a substance or the greater its capacity to substitute. It is, therefore, reactive and is termed activated . Activated phosphates are, for example, acyl phosphates and acyl AMP derivatives (C 1.2), sugar phosphates (C 6), and isopentenyl pyrophosphate (D 6). 

Phosphotransferases catalyze the transfer of a phosphate group from one compound to another according to the following equation  

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FIGURE 10.26 Glucose transport in E. coli is mediated by the PEP-dependent phosphotransferase system. Enzyme I is phosphorylated in the first step by PEP. Successive phosphoryl transfers to HPr and Enzyme III in Steps 2 and 3 are followed by transport and phosphorylation of glucose. Enzyme II is the sugar transport channel. 

Several unique features distinguish the phosphotransferase. First, phos-phoenolpyruvate is both the phosphoryl donor and the energy source for sugar transport. Second, four different proteins are required for this transport. Two of these proteins (Enzyme I and HPr) are general and are required for the phosphorylation of all PTS-transported sugars. The other two proteins (Enzyme II and Enzyme III) are specific for the particular sugar to be transported. 

In this chapter, we have examined coupled transport systems that rely on ATP hydrolysis, on primary gradients of Na or Ff, and on phosphotransferase systems. Suppose you have just discovered an unusual strain of bacteria that transports rhamnose across its plasma membrane. Suggest experiments that would test whether it was linked to any of these other transport systems. 

Meadow, N. D., Fox, D. K., and Roseman, S., 1990. The bacterial phos-phoenolpyrnvate glycose phosphotransferase system. Annual Review of Biochemistry 59 497-542. 

T"he extraordinary ability of an enzyme to catalyze only one particular reaction is a quality known as specificity (Chapter 14). Specificity means an enzyme acts only on a specific substance, its substrate, invariably transforming it into a specific product. That is, an enzyme binds only certain compounds, and then, only a specific reaction ensues. Some enzymes show absolute specificity, catalyzing the transformation of only one specific substrate to yield a unique product. Other enzymes carry out a particular reaction but act on a class of compounds. For example, hexokinase (ATP hexose-6-phosphotransferase) will carry out the ATP-dependent phosphorylation of a number of hexoses at the 6-posi-tion, including glucose. 

The transport of each COg requires the expenditure of two high-energy phosphate bonds. The energy of these bonds is expended in the phosphorylation of pyruvate to PEP (phosphoenolpyruvate) by the plant enzyme pyruvate-Pj dikinase the products are PEP, AMP, and pyrophosphate (PPi). This represents a unique phosphotransferase reaction in that both the /3- and y-phosphates of a single ATP are used to phosphorylate the two substrates, pyruvate and Pj. The reaction mechanism involves an enzyme phosphohistidine intermediate. The y-phosphate of ATP is transferred to Pj, whereas formation of E-His-P occurs by addition of the /3-phosphate from ATP ... 

Beside AAC enzymes two different enzyme classes, nucleotidyltransferases (ANT enzymes), and phosphotransferases (APH enzymes) modify the hydroxyl groups of aminocyclitol-aminoglycoside antibiotics. 

O-phosphotransferases that modify macrolides are produced by highly macrolide resistant E. coli isolates. However, these enzymes have no clinical importance for macrolide resistance in gram-positive bacteria, and gram-negative ones are regarded as naturally resistant [2]. 

Disorders caused by mislocalization of lysosomal proteins (e.g., mutations in the N-acetylglucosamine 1-phosphotransferase leading to inclusion cell disease). 

ATP interacts with APS to form PAPS, catalyzed by ATP adenylyl sulfate 3-phosphotransferase. 

An important additional pathway is indicated in reactions I and II of Figure 47-9. This involves enzymes destined for lysosomes. Such enzymes are targeted to the lysosomes by a specific chemical marker. In reaction I, a residue of GIcNAc-1-P is added to carbon 6 of one or more specific Man residues of these enzymes. The reaction is catalyzed by a GIcNAc phosphotransferase, which uses UDP-GlcNAc as the donor and generates UMP as the other product ... 

Man 6-P receptors, located in the Golgi apparatus, bind the Man 6-P residue of these enzymes and direct them to the lysosomes. Fibroblasts from patients with I-cell disease (see below) are severely deficient in the activity of the GIcNAc phosphotransferase. 

Pseudo-Hurler polydystrophy is another genetic disease closely related to I-ceU disease. It is a milder condition, and patients may survive to adulthood. Smdies have revealed that the GlcNAc phosphotransferase involved in I-cell disease has several domains, including a catalytic domain and a domain that specifi-... 

The Enzymes II (E-IIs) of the phosphoenolpyruvate (P-enolpyruvate)-dependent phosphotransferase system (PTS) are carbohydrate transporters found only in prokaryotes. They not only transport hexoses and hexitols, but also pentitols and disaccharides. The PTS substrates are listed in Table I. The abbreviations used (as superscripts) throughout the text for these substrates are as follows Bgl, jS-gluco-side Cel, cellobiose Fru, fructose Glc, glucose Gut, glucitol Lac, lactose Man, mannose Mtl, mannitol Nag, iV-acetylglucosamine Scr, sucrose Sor, sorbose Xtl, xylitol. 

Larsson, R. and Cerutti, P. (1989). Translocation and enhancement of phosphotransferase activity of protein kinase C following exposure of mouse epidermal cells to oxidants. Cancer Res. 49, 5627-5632. 

Maximum residue limit Mass spectrometry Tandem mass spectrometry Material safety data sheet North American Free Trade Act N-Hydroxysuccinimide Nitrogen-phosphorus detection Neomycin phosphotransferase II Optical density Office of Plant Protection and Quarantine... 

Neomycin phosphotransferase II (NPTII) extraction from cotton leaves and cottonseed. The extraction buffer consists of 100 mM Tris, lOmM sodium borate, 5mM magnesium chloride, 0.2% ascorbate and 0.05% Tween 20 at pH 7.8. The frozen leaf sample is homogenized in cold (4 °C) buffer. An aliquot of the homogenate is transferred to a microfuge tube and centrifuged at 12 000 g for 15 min. The supernatant is diluted and assayed directly by ELISA. 

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