Nonenzymatic reactions

The question of which odorants are formed in which amounts when food is heated depends on the usual parameters of a chemical reaction. These are the chemical structure and concentration of the precursors, temperature, time and environment, e. g., pH value, entry of oxygen and the water content. Whether the amounts formed are really sufficient for the volatiles to assert themselves in the aroma depend on their odor thresholds and on interactions with other odorants. 

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Cyanohydrin Synthesis. Another synthetically useful enzyme that catalyzes carbon—carbon bond formation is oxynitnlase (EC 4.1.2.10). This enzyme catalyzes the addition of cyanides to various aldehydes that may come either in the form of hydrogen cyanide or acetone cyanohydrin (152—158) (Fig. 7). The reaction constitutes a convenient route for the preparation of a-hydroxy acids and P-amino alcohols. Acetone cyanohydrin [75-86-5] can also be used as the cyanide carrier, and is considered to be superior since it does not involve hazardous gaseous HCN and also virtually eliminates the spontaneous nonenzymatic reaction. (R)-oxynitrilase accepts aromatic (97a,b), straight- (97c,e), and branched-chain aUphatic aldehydes, converting them to (R)-cyanohydrins in very good yields and high enantiomeric purity (Table 10). 

Enzymatic methylation of homocysteine (HSCHjCHjCHNHjCOOH) by methylcobalamin to give methionine (CH3SCH2CH2CHNH2COOH) was discovered in 1962 by Woods and co-workers, who also noticed the occurrence of a much slower, nonenzymatic reaction giving the same products. Methylcobinamide showed the same activity as the cobalamin in both the enzymatic and nonenzymatic reactions (72, 7/). It was subsequently discovered that HS, MeS , PhS , and w-BuS will dealkylate a variety of methyl complexes [DMG, DMG-BF2, DPG, G, salen, (DO)(DOH)pn, cobalamin] and even ethyl-Co(DMG)2 complexes to give the thioethers, and it was suggested that the reaction involved transfer of the carbonium ion to the attacking thiolate 161, 164), e.g.,... 

Oberhuber, M. et al.. Breakdown of chlorophyll A nonenzymatic reaction accounts for the formation of the colorless nonfluorescent chlorophyll catabolites, PNAS, 100, 6910, 2003. 

Dopachrome also undergoes a nonenzymatic reaction to form dihidroxyindole (DHI), the precursor of DHI-eumelanins. For the formation of phaeomelanins, dopaquinone is first transformed in cysteinil-DOPA and then in cysteinyl-dopaquinone which suffers a nonenzymatic polymerization. The polymerization of monomers and the association of melanins with proteins is not yet completely elucidated and may involve other intermediates. ... 

At low temperatures, the nonenzymatic reaction is reduced to a larger extent than the enzymatic reaction. The mass transfer rate is reduced to a smaller extent. Mass transfer limitation is required for high enantiomeric excess and determines the conversion rate. Therefore, the volumetric productivity decreases at lower temperatures. The equilibrium constant is considerably higher at low temperatures, resulting in a higher extent of conversion or a lower HCN requirement. Both the volumetric productivity and the required enzyme concentration increase by increasing the reaction temperature and aqueous-phase volume while meeting the required conversion and enantiomeric excess [44]. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalize and predict [45],... 

The process by which a xenobiotic is structurally and/or chemically changed in the body by either enzymatic or nonenzymatic reactions. The product of the reaction is a different composition of matter or different configuration than the original compound. 

Remote double labelling techniques have been used successfully in the determination of enzyme KIEs (Kiick, 1991). A variant of this technique was applied to a nonenzymatic reaction by Matsson and co-workers (Axelsson et al., 1990). They determined the primary carbon and secondary deuterium KIEs for the SN2 reaction between methyl iodide and hydroxide ion in 50% dioxane-water at 25°C. The a-carbon KIE was determined by the UC method (Axelsson et al. 1987,1991). In this method, a mixture of substrate molecules labelled with UC (tm = 20.4 min) and 14C is used. The reactants and products... 

Prodrug activation occurs enzymatically, nonenzymatically, or, also, sequentially (an enzymatic step followed by a nonenzymatic rearrangement). As much as possible, it is desirable to reduce biological variability, hence the particular interest currently received by nonenzymatic reactions of hydrolysis or intramolecular catalysis [18][20], Reactions of cyclization-elimination appear quite promising and are being explored in a number of studies. 

The relative contribution of the enzymatic and nonenzymatic reactions is frequently examined but seldom reported explicitly in in vitro studies, and is all but impossible to assess in in vivo investigations. Another factor complicating the interpretation of in vivo results is the contribution of gastrointestinal microflora, whose hydrolytic capacity is particularly marked and varied... 

In summary, (oxodioxolyl)methyl esters of carboxylic acid drugs appear to be generally useful as prodrugs. However, more studies are needed to document the structure-metabolism relationships, the relative contribution of enzymatic vs. nonenzymatic reactions in their in vivo activation, the reasons of some failures, their toxic potential, and their pharmacokinetic behavior in humans. 

B. Ketterer, The Role of Nonenzymatic Reactions of Glutathione in Xenobiotic Metabolism , Drug Metab. Rev. 1982, 13, 161 - 187 B. Ketterer, G. J. Mulder, Glutathione Conjugation , in Conjugations Reactions in Drug Metabolism , Ed. G. J. Mulder, Taylor and Francis, London, 1990, p. 307 - 364. 

The relevance of this mechanism to mammalian enzymes is an important question, but we are not aware of any detailed study that affords a definitive answer. Proof that reactions of hydrolytic dehalogenation ofhaloalkyl groups occur in animals is presented in the next subsection, but much remains to be discovered regarding the enzymes involved or the reaction mechanisms. Furthermore, nonenzymatic reactions remain a distinct possibility when the C-atom bearing the halogen is sufficiently electrophilic, as seen, e.g., with (2-chloroethyl)amino derivatives (see Sect. 11.4.2). 

Even lower temperatures have been used to study possible intermediate stages in the formation of the acyl enzyme. A tetrahedral intermediate (with a covalent bond between the substrate carbonyl carbon atom and the oxygen atom of the active site serine) (Fig. 2) had been suggested by analogy with nonenzymatic reactions. With rapid reaction techniques, spectrophotometric evidence has been obtained for an additional intermediate before the acyl enzyme in the case of chromophoric substrates. By using first the protein fluorescence emission (Fink and Wildi, 1974)... 

Hydroxymethylglutaryl-CoA lyase 4.1.3.4 3-Hydroxybutyrate dehydrogenase 1.1.1.30 Nonenzymatic reaction... 

In resting muscle, creatine phosphate forms due to the high level of ATP. If there is a risk of a severe drop in the ATP level during contraction, the level can be maintained for a short time by synthesis of ATP from creatine phosphate and ADP. In a nonenzymatic reaction [6], small amounts of creatine and creatine phosphate cyclize constantly to form creatinine, which can no longer be phosphorylated and is therefore excreted with the urine (see p. 324). 

INFLUENCE OF OTHER STEPS ON THE MAGNITUDE OF OBSERVED ISOTOPE EFFECTS. As noted earlier, nonenzymatic reaction mechanisms do not involve those complexities imposed by substrate binding order, rates of substrate binding/release, as well as conformational changes that attend enzyme catalysis. As a result, the opportunity for detecting isotope effects is... 

When it is feasible to identify and characterize a single rate process, the effect of pH is more straightforward. This is frequently the case for nonenzymatic reactions obeying simple rate equations, but fast reaction techniques sometimes allow one to examine a single elementary reaction in an enzymic process. In either case, the behavior shown in Fig. 3 typifies the manner in which an observed reaction rate constant depends on the pT a of an ionizable group. 

In a number of nonenzymatic reactions catalyzed by pyridoxal, a metal ion complex is formed—a combination of a multivalent metal ion such as cupric oi aluminum ion with the Schiff base formed from the combination of an amino acid and pyridoxal (I). The electrostatic effect of the metal ion, as well as the electron sink of the pyridinium ion, facilitates the removal of an a -hydrogen atom to form the tautomeric Schiff base, II. Schiff base II is capable of a number of reactions characteristic of pyridoxal systems. Since the former asymmetric center of the amino acid has lost its asymmetry, donation of a proton to that center followed by hydrolytic cleavage of the system will result in racemic amino acid. On the other hand, donation of a proton to the benzylic carbon atom followed by hydrolytic cleavage of the system will result in a transamination reaction—that is, the amino acid will be converted to a keto acid and pyridoxal will be converted to pyridoxamine. Decarboxylation of the original amino acid can occur instead of the initial loss of a proton. In either case, a pair of electrons must be absorbed by the pyridoxal system, and in each case, the electrostatic effect of the metal ion facilitates this electron movement, as well as the subsequent hydrolytic cleavage (40, 43). 

Fortunately, data have been collected by Speck (41) on the activity of a large number of metals in the enzymatic as well as the nonenzymatic reaction. 

The most remarkable feature that can be gleaned from the examination of Figure 4 is that the order of metal activation of the nonenzymatic reaction follows generally the order of complex stability, as Williams has pointed out (52). This is particularly true at low metal ion concentration—e.g., 10-3 M—where the metals in the first transition series, for example, are active in the order Mn+2Zn+2, the well-known Irving-Williams (25) order of complex stability. The other metals also fall in line. 

Recently, Senoh, Tokuyama, and Witkop (37) have studied a metal-activated enzymatic reaction in the presence and the absence of enzyme, and have discovered that the order of effectiveness of the metals is exactly the reverse in the enzymatic and nonenzymatic processes. The reaction was O-methylation of 3,4-dihydroxybenzaldehyde. In the absence of divalent metal ions, the nonenzymatic reaction yields very predominantly the paramethylated product in neutral solution, since the p-hydroxyl group is the more electronegative. Metal complex formation... 

Lowenstein reacted ATP with orthophosphate (33) in the presence of metal ions, and obtained ADP and pyrophosphate as products. The most active metals in this reaction were, rather surprisingly for a nonenzymatic reaction, the alkaline earths, Cd+2 and Mn+2 the members of the first transition series exhibited low7 activity. The reactive intermediate was formulated as follows ... 

The enzymatic reaction was postulated to involve the same intermediate, with some of the coordinate bonds of the metal also attached to ligands on the enzyme. It was also possible, in the nonenzymatic reaction, to substitute an organic acid, such as acetate (34) for the orthophosphate the reaction proceeded in like fashion to produce a phosphate ester ... 

RGURE 8-33 Some well-characterized nonenzymatic reactions of nucleotides (a) Deamination reactions Only the base is shown, (b) Depurination, in which a purine is lost by hydrolya s of the N-fS-glycosyl bond. The deoxyribose remaining after depurination is readily converted from the /3-furanose to the aldehyde form (see Rg. 8-3). Further nonenzymatic reactions are illustrated in figures 8-34 and 8-35. 

In the nonenzymatic reaction of Eq. 9-76 the ionic atmosphere provided by positive counterions in solution can continuously readjust to keep the negative charge effectively balanced at every step along the reaction coordinate and through the transition state. Within enzymes this adjustment may occur via redistribution of electrical charges within the polarizable network of internal hydrogen bonds. The enzyme structure must allow this. Because of the complexity of an enzymatic transition state it may be hard to compare it with the transition state of the corresponding nonenzymatic... 

The fact that enzymes appear to bind their substrates in such a way as to surround and immobilize them means that something other than the kinetic energy of the substrate is needed to provide energy for the ES complex to pass over the transition state barrier. What is the source of this activation energy As with nonenzymatic reactions, it must come ultimately from... 

Figure 12-21 illustrates the use of the technique in investigating the possible participation of metaphosphate in a nonenzymatic reaction. 

Pyridoxal or PLP, in the complete absence of enzymes, not only undergoes slow transamination with amino acids but also catalyzes many other reactions of amino acids that are identical to those catalyzed by PLP-dependent enzymes. Thus, the coenzyme itself can be regarded as the active site of the enzymes and can be studied in nonenzymatic reactions. The latter can be thought of as models for corresponding enzymatic reactions. From such studies Snell and associates drew the following conclusions.148... 

Many investigations of nonenzymatic reactions of PLP and related compounds have been and are still being conducted149 150... 

PQQ and the other quinone prosthetic groups described here all function in reactions that would be possible for pyridine nucleotide or flavin coenzymes. All of them, like the flavins, can exist in oxidized, half-reduced semiquinone and fully reduced dihydro forms. The questions to be asked are the same as we asked for flavins. How do the substrates react How is the reduced cofactor reoxidized In nonenzymatic reactions alcohols, amines, and enolate anions all add at C-5 of PQQ to give adducts such as that shown for methanol in Eq. 15-51, step a 444,449,449a Although many additional reactions are possible, this addition is a reasonable first step in the mechanism shown in Eq. 15-51. An enzymatic base could remove a proton as is indicated in step b to give PQQH2. The pathway for reoxidation (step c) might involve a cytochrome b, cytochrome c, or bound ubiquinone.445 446... 

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