Phosphorylating ability

The alkyl and alkoxy substituents of phosphate or phosphonate esters also affect the phosphorylating ability of the compound through steric and inductive effects. A satisfactory correlation has been developed between the quantitative measure of these effects, Tafts s (7, and anticholinesterase activity as well as toxicity (33). Thus long-chain and highly branched alkyl and alkoxy groups attached to phosphorus promote high stability and low biological activity. 

Szabo (1967) found that, when the two alkyl radicals of the phosphonic esters are different, the compounds have a stronger insecticidal action than in the case of identical alkyl radicals. This is in agreement with a similar finding of Melnikov (1963) in relation to parathion derivatives. This fenomenon is evidently due to the fact that asymmetry increases the polarisation of the central phosphorus atom, resulting in an increased phosphorylating ability. 

Some of the regulatory enzymes involved in the phosphorylation/dephosphorylation of helminth glycolytic enzymes have been characterized. Two distinct protein kinases have been purified from A. suum muscle (25,26). One is cAMP-dependent and appears to be similar to the corresponding mammalian enzyme. Its apparent for PFK is similar to the intracellular concentration of the PFK, suggesting that PFK may be a substrate in vivo. The second kinase activity is cAMP-independent and not affected by the A. suum cAMP binding protein, Walsh inhibitor, ox heart R subunit or antiserum to the ox heart cAMP-dependent protein kinase (25). This kinase phosphorylates histone, phosvitin, and the A. suum PFK and phosphorylase b. It is responsible for about 75% of the PFK phosphorylating ability in A. suum muscle homogenates. 

This demonstration of a decreased phosphorylating ability and increased ATPase activity of mitochondria from thyrotoxic animals cannot be taken as evidence that the hormone directly affects these enzymes. Indeed, in both instances it appears that the changes are secondary to a primary effect on the mitochondria themselves (see below). These apparent effects of the thyroid hormones on oxidative phosphorylation and ATPase activity have been duplicated in vitro, and will be discussed below. 

The three sites of phosphorylation suspected to exist in intact mitochondria have also been recognized in the phosphorylating particle where these sites do not function at full capacity. Although the first of these sites (between NAD and the flavoprotein) operates at 90% of its full capacity, the second (between cytochrome c and cytochrome c ) is active at only 20% of its normal phosphorylating ability, and the third (between cytochrome c and oxygen) reaches 70% of the normal phosphorylating rates. 

Some of the chemical properties of the phosphorylating particle have also been elucidated. The particle consists mainly of two basic components, protein (65%) and phospholipids (20%). In addition to the various components of the electron transport chain, the phosphorylating particle also contains ATPase, the activity of which is so high that it may well explain the particle s low phosphorylating ability. Like that associated with mitochondria, the ATPase found in the particle is a highly specific enzyme that splits ATP to yield ADP, and is without effect on inosine, guano-sine, or cytidine triphosphate. There is more bound copper and iron in these particles than can be accounted for by the ferroproteins and cuproproteins of the electron transport chain. 

Lehninger s studies indicate that thyroxine affects mitochondrial swelling. These studies do not yet exclude the possibility that the hormone acts directly at one or more phosphorylation sites. An effect of thyroxine on the oxidative phosphorylation abilities of mitochondrial fragments obtained by sonication of mitochondria has been demonstrated thyroxine and triiodothyronine stimulate the oxidation of succinate and NAD and enhance the rate of phosphorylation. In contrast, tetraiodothyroacetic acid and triiodothy-roacetic acid uncouple oxidative phosphorylation. 

The biochemical basis for the toxicity of mercury and mercury compounds results from its ability to form covalent bonds readily with sulfur. Prior to reaction with sulfur, however, the mercury must be metabolized to the divalent cation. When the sulfur is in the form of a sulfhydryl (— SH) group, divalent mercury replaces the hydrogen atom to form mercaptides, X—Hg— SR and Hg(SR)2, where X is an electronegative radical and R is protein (36). Sulfhydryl compounds are called mercaptans because of their ability to capture mercury. Even in low concentrations divalent mercury is capable of inactivating sulfhydryl enzymes and thus causes interference with cellular metaboHsm and function (31—34). Mercury also combines with other ligands of physiological importance such as phosphoryl, carboxyl, amide, and amine groups. It is unclear whether these latter interactions contribute to its toxicity (31,36). 

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. 

It s this ability to drive otherwise unfavorable phosphorylation reactions that makes ATP so useful. The resultant phosphates are much more reactive as leaving groups in nucleophilic substitutions and eliminations than the corresponding alcohols they re derived from and are therefore more likely to be chemically useful. 

Microorganisms under anaerobic growth conditions have the ability to utilise glucose by the Embden-Mereyhof-Parnas pathway.4 Carbohydrates are phosphorylated through the metabolic pathway the end products are two moles of ethanol and carbon dioxide.5... 

We have studied the extractant behavior of a series of compounds containing the carbamoylmethylphosphoryl (CMP) moiety in which the basicity of the phosphoryl group and the steric bulk of the substituent group are varied (10,LL). These studies have led to the development of extractants which have combinations of substituent groups that impart to the resultant molecule improved ability to extract Am(III) from nitric acid and to withstand hydrolytic degradation. At the same time good selectivity of actinides over most fission products and favorable solubility properties on actinide loading are maintained (11). 

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and ph osphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases. 

Fig. 4. Domain complementation schemes. (A) A domain complementation. The H554A site-directed mutant is inactive in P-enolpyruvate-dependent mannitol phosphorylation because it cannot accept a phosphoryl group from P-FIpr. The measure of A domain activity is its ability to restore mannitol phosphorylation activity to this mutant. A domain activity in the AB subcloned protein can also be measured. (B) B domain complementation. The C384S site-directed mutant is inactive in P-enolpyruvate-dependent mannitol phosphorylation because it cannot pass the phosphoryl group from H554 on its own A domain to mannitol. The measure of B domain activity is its ability to restore mannitol phosphorylation activity to this mutant. B domain activity in the AB subcloned protein can also be measured. (C) C domain complementation. The activity of the C domain is measured by complementation with the purified AB domain.

Dinitrophenol is a member of the aromatic family of pesticides, many of which exhibit insecticide and fungicide activity. DNP is considered to be highly toxic to humans, with a lethal oral dose of 14 to 43mg/kg. Environmental exposure to DNP occurs primarily from pesticide runoff to water. DNP is used as a pesticide, wood preservative, and in the manufacture of dyes. DNP is an uncoupler, or has the ability to separate the flow of electrons and the pumping of ions for ATP synthesis. This means that the energy from electron transfer cannot be used for ATP synthesis [75,77]. The mechanism of action of DNP is believed to inhibit the formation of ATP by uncoupling oxidative phosphorylation. 

Freeman, R. S., Meyer, A. N., Li, J., and Donoghue, D. J. (1992). Phosphorylation of conserved serine residues does not regulate the ability of mas" protein kinase to induce meiotic maturation or function as cytostatic factor. J. Cell Biol. 116 725-735. 

---