Biological systems phosphates

C. J. Anderson, L. J. H. Lucas, T. S. Widlanski, Molecular Recognition in Biological Systems Phosphate Esters vs. Sulfate Esters and the Mechanism of Action of Steroid Sulfatases , J. Am. Chem. Soc. 1995,117, 3889-3890. 

The biological utilization of phosphate derivatives is of utmost importance. Phosphate diesters form part of the backbone of DNA. The phosphate derivatives and anhydrides, in particular ATP, are the main method of energy transduction in the cell and phosphorylation of proteins is a key step in the regulation of some metabolic pathways. In contrast to their reactivity in biological systems, phosphate derivatives are generally un-reactive in laboratory conditions. A striking feature of many of the enzymes in this area is that most of them are metalloenzymes or require metal ion cofactors (108). This observation prompts the question. Do metal ions have a special effect on the reactivity of phosphate derivatives or are they merely used in a structural role (140) A number of mechanisms by which metal ions could conceivably facilitate the reactions of phosphate derivatives are fairly obvious and include the six modes listed below. 

Phase I of the toxicological study was undertaken to determine whether or not some of our initial assumptions (Focal Points) were correct in biological systems. Phosphate fibers were studied in biological media similar to those encountered in living organisms.Rates of solution, hydrolytic degradation, and relative toxicity were determined in some very elegant and definitive experiments. The physical chemical studies were probably some of the more reliable ever reported. Fortunately there were no surprises. 

So far, as in Equation (3.33), the hydrolyses of ATP and other high-energy phosphates have been portrayed as simple processes. The situation in a real biological system is far more complex, owing to the operation of several ionic equilibria. First, ATP, ADP, and the other species in Table 3.3 can exist in several different ionization states that must be accounted for in any quantitative analysis. Second, phosphate compounds bind a variety of divalent and monovalent cations with substantial affinity, and the various metal complexes must also be considered in such analyses. Consideration of these special cases makes the quantitative analysis far more realistic. The importance of these multiple equilibria in group transfer reactions is illustrated for the hydrolysis of ATP, but the principles and methods presented are general and can be applied to any similar hydrolysis reaction. 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

Tu, A. J., Heller, M. J. Structure and Stability of Metal-Nucleoside Phosphate Complexes, in Metal Ions in Biological Systems Vol. 1 (ed. Sigel, H.), p. 1, Marcel Dekker, Inc. New York 1974... 

Electronically, we find that strongly polarized acyl compounds react more readily than less polar ones. Thus, acid chlorides are the most reactive because the electronegative chlorine atom withdraws electrons from the carbonyl carbon, whereas amides are the least reactive. Although subtle, electrostatic potential maps of various carboxylic add derivatives indicate the differences by the relative blueness on the C-O carbons. Acyl phosphates are hard to place on this scale because they are not used in the laboratory, but in biological systems they appear to be somewhat more reactive than thioesters. 

SCHEME 10.2 Common pathways of QM formation in biological systems, (a) Stepwise two-electron oxidation by cytochrome P450 or a peroxidase, (b) Enzymatic oxidation of a catechol followed by spontaneous isomerization of the resulting n-quinone. (c) Enzymatic hydrolysis of a phosphate ester followed by base-catalyzed elimination of a leaving group from the benzylic position. 

DNA and RNA are formed of nucleotides. Each nucleotide or nucleoside is composed of a purine or pyrimidine base linked to the 1-position of a ribose sugar in the case of RNA and a 2 -deoxyribose sugar in the case of DNA.155 The 5 position is phosphorylated in the case of a nucleotide, while the nucleoside is not phosphorylated therefore, nucleotides are nucleoside phosphates. Phosphorylation can include one, two, or three phosphate groups. The most familiar example of a phosphorylated nucleotide is phosphorylated adenosine, which occurs as the mono-, di-, and triphosphate (AMP, ADP, and ATP, respectively) and is a principal means of energy storage in biological systems. 

Fig. 2.7. Characteristic rate constants (s 1) for substitution of inner-sphere H20 of various aqua ions. Note The substitution rates of water in complexes ML(H20)m will also depend on the symmetry of the complex (adapted from Frey, C.M. and Stuehr, J. (1974). Kinetics of metal ion interactions with nucleotides and base free phosphates in H. Sigel (ed.), Metal ions in biological systems (Vol. 1). Marcel Dekker, New York, p. 69).

Another reason for neglecting perhydroxyl radical is a big difficulty to distinguish it from the much more abundant and more reactive peroxyl radicals. Nonetheless, in several works perhydroxyl radical was considered as a possible initiator of lipid peroxidation (see Chapter 25). It should be noted that at least two biological systems were described where the participation of perhydroxyl radicals seems to be possible. Thus, it has been shown [25,26] that perhydroxyl radical is able to abstract hydrogen atom from NADH (Reaction 6) and the glyceraldehyde-3-phosphate dehydrogenase-NADH (GAPDH-NADH) complex (Reaction 7). 

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

Similarly, specific catalysts called enzymes are important factors in determining what reactions occur at an appreciable rate in biological systems. For example, adenosine triphosphate is thermodynamically unstable in aqueous solution with respect to hydrolysis to adenosine diphosphate and inorganic phosphate. Yet this reaction proceeds very slowly in the absence of the specific enzyme adenosine triphosphatase. This combination of thermodynamic control of direction and enzyme control of rate makes possible the finely balanced system that is a hving cell. 

A similar situation is found in the structure of putrescine diphosphate " (a model system for amine-nucleic acid interactions) which divides into layers of HjPOJ anions bridged by protonated putrescine (1,4-diamino-n-butane) cations. In a real biological system (yeast phenylalanine transfer RNA) phosphate residues are found to be enveloped by the polyamine spermine [NH2(CH2)jNH(CH2)4NH(CH2)jNH2] which again adopts a linear, nonchelating conformation. ... 

Monosaccharides also form phosphate esters with phosphoric acid. Monosaccharide phosphate esters are important molecules in biological system. For example, in the DNA and RNA nucleotides, phosphate esters of 2-deoxyribose and ribose are present, respectively. Adenosine triphosphate (ATP), the triphosphate ester at C-5 of ribose in adenosine, is found extensively in living systems. 

Although the metaphosphate mechanism for hydrolysis is well documented, such a pathway remains to be demonstrated in a biological system. Our present knowledge of many enzymic reactions allows, at best, the formulation of a preliminary mechanism, i.e. the chemical identity of substrates and enzymic intermediates and the minimal kinetic scheme. For example, much recent attention has been focused on the remarkable stability of the covalent phos-phoryl-enzyme (an O-phosphoryl serine) derived from E. coii alkaline phosphatase28 and inorganic phosphate, and on a systematic kinetic study of the enzyme s substrate specificity (O-, N- and S-monoesters) -9. Dephosphorylation of the enzyme, however, does not appear to be via a metaphosphate mechanism30. 

The above discussion has relied to a considerable extent on the concepts developed in the chemistry of cyclic phosphates. Obviously pentacovalent species are not required on the basis of the available experimental evidence in many of the above reactions and alternate explanations are plausible. Nevertheless, many diverse observations are explained with equal facility by application of these concepts. Although this certainly does not constitute a proof, the approach offers a unifying framework for productive experimental design with both chemical and biological systems. One relevant example s the previously... 

Mechanisms may be readily envisaged where this reaction may lead to phos-phoryl transfer through nucleophilic attack with ring opening on phosphorus followed by hydrolysis of the acyclic acyl phosphate. In biological systems this may function as an alternative to the metaphosphate reaction. 

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