Aldose reductase: physiological role, properties and prospects for regulating activity]

Some properties of aldose reductase isolated from various sources and possible ways of regulation of the enzyme catalytic activity are reviewed. Mammalian aldose reductases are monomeric enzymes with M(r) of 30-40 kDa and a broad substrate specificity towards aldoses. The physiological role of this enzyme consists, apparently, in providing an additional pathway for utilization of glucose and removing toxic compounds carrying an aldehyde group from the cell. Aldose reductase is thought to play a key role in various hyperglycemic states, including diabetic cataract. The kinetics of the aldose reductase reaction is hyperbolic with NADPH and nonhyperbolic with glucose. The rate of the enzyme-catalyzed reaction is determined by the effector binding in the active of inhibitory center of the enzyme. Incubation with substrates leads to the activation of the enzyme which is accompanied by a decrease of the effector binding in the enzyme inhibitory center with a sharp decrease in the sensitivity of the activated enzyme to NADPH concentration changes in the presence of glucose excess. A mechanism underlying the catalytic effect of both native and activated forms of the enzyme is proposed.     

N-nitrosamines: bacterial mutagenesis and in vitro metabolism

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The kinetics of chemical reaction of activated nitrosamine with organic substrates.

The chemical reaction of tosylated N-nitrosobis (2-hydroxylpropyl) amine with aniline, 1-naphthol, pyridine, and hydroxylamine follows kinetics which can be described in a mixed order rate equation. This nitrosamine, considered an activated nitrosamine with a potential for alkylating a substrate, was seen to alkylate aniline, pyridine, 1-naphthol, and hydroxylamine. The presence of salts in the solution as well as an increase in solution pH increased the rate of reaction of the nitrosamine with substrates. The nitrosamine reacts quickly with water but will not react with solvents such as acetonitrile, dimethylsulfoxide, and methylene chloride.     

Activation of cerebral guanylate cyclase by nitric oxide.

Mouse cerebral guanylate cyclase was activated by catalase in the presence of sodium azide (NaN₃), which is known to form catalase-NO complex, while nitrosamines and nitric oxide (NO gas) were capable of activating cerebral guanylate cyclase in the absence of catalase. The activation of guanylate cyclase by NaN₃-catalase or nitrosamines was markedly inhibited by ferrohemoglobin which has a high affinity for NO, but not by ferrihemoglobin. These data suggest that NO or NO containing compounds may activate guanylate cyclase, whereas ferrohemoglobin may exhibit an inhibitory effect on the activation of guanylate cyclase, possibly by interacting with NO or NO containing compounds.     

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.     

New substrates for beta-lactam-recognizing enzymes: aryl malonamates

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.     

7-azabicyclo[2.2.1]heptane as a structural motif to block mutagenicity of nitrosamines

Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.     

Stereochemical effects in the metabolic activation of nitrosopiperidines: correlations with genotoxicity

The genotoxicity of nitrosopiperidine and six alkyl derivatives was studied by use of the Ames tester strains TA100 and TA1535 in the pre-incubation method. Among the compounds investigated, those exhibiting genotoxic activity under the experimental conditions employed were only genotoxic in the presence of S9 mix (10%). The results obtained were correlated with models of the metabolic activation of nitrosamines in an attempt to rationalize the genotoxicity of these compounds. The results show that the presence of substituents in the nitrosopiperidine molecule may be one of the modulating factors affecting the genotoxicity of these cyclic nitrosamines, and may help provide some chemical clues for the identification of risk compounds from among a large group of structurally related molecules.     

Mechanisms of inhibition of N-nitroso compounds-induced mutagenicity

In this review we describe the mechanisms of the inhibitory effects of various chemical agents towards the mutagenicity of N-nitroso compounds, including direct-acting mutagens such as N-nitroso derivatives of alkylureas, alkylnitroguanidines and alkylurethanes, and promutagenic nitrosamines. Possible mechanisms by which the inhibitors may exert their effects outside and inside the target cells include chemical and enzymatic deactivation of the mutagen, inhibition of metabolic activation of nitrosamines, scavenging mutagenic products, inhibition of cellular uptake, induction of detoxifying mechanisms, protecting nucleophilic centers in DNA and modulating DNA repair.     

Lipases in lipophilization reactions

Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives.     

Deuterium isotope effects in mutagenesis by nitroso compounds.

Nitrosamines which have deuterium instead of hydrogen in the position alpha to the nitroso group have been reported to have reduced activity in carcinogenicity tests. This result implies that cleavage of a carbon--hydrogen bond is a limiting step in the reaction mechanism leading to tumor formation. Mutagenicity tests were undertaken with nitrosamines, which require metabolic activation, and with nitrosamides, which are directly acting mutagens, to determine the effect of deuterium substitution on the activity of each type of compound. Two nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and methylnitrosourea) and three nitrosamines (dimethylnitrosamine, nitrosomorpholine, and dinitrosopiperazine) and their deuterium-containing analogs were tested for reversion of a nonsense mutation in the tyr locus of Escherichia coli WU 3610 (tyr-, leu-). Nitrosamines activated by rat-liver microsomes, but not nitrosamides, were less active as mutagens when the deuterium atom was present. The results suggest that the metabolic activation of nitrosamines to a mutagenic species involves the loss of hydrogen, a reaction which the nitrosamides, in the absence of enzyme, do not undergo.     

Kinetics of phenol biodegradation in the presence of glucose

The kinetics of utilization of glucose, phenol, and their mixtures by Pseudomonas putida (ATCC 17514) were studied with a continuously aerated, jacketed batch reactor operating at 28 degrees C and pH 7.2. It was found that when glucose is the sole carbon and energy source, the culture utilizes it following Monod kinetics. When phenol is the sole carbon and energy source, the culture biodegrades it following Andrews (inhibitory) kinetics. When both glucose and phenol are present in the medium, the culture uses them simultaneously but with lower specific rates. Reduction of the specific substrate utilization rates indicates that the two substances are involved in a cross-inhibitory pattern which can be classified as uncompetitive. The values of the kinetic interaction constants suggest that glucose inhibits the specific rate of phenol removal much more than phenol inhibits the specific rate of glucose utilization. The results suggest that substitutable substrates which are dissimilar in origin and molecular structure may be involved in an uncompetitive cross-inhibitory interaction when they are simultaneously removed. It is also concluded that the use of easily degradable substrates may not enhance the per-unit amount of biomass removal of compounds which are classified as toxic. A general classification of kinetic interactions between substitutable resources is proposed. (c) 1996 John Wiley & Sons, Inc.     

A ribonucleotide Origin for Life – Fluctuation and Near-ideal Reactions

Oligoribonucleotides are potentially capable of Darwinian evolution – they may replicate and can express an independent chemical phenotype, as embodied in modern enzymatic cofactors. Using quantitative chemical kinetics on a sporadically fed ribonucleotide pool, unreliable supplies of unstable activated ribonucleotides A and B at low concentrations recurrently yield a replicating AB polymer with a potential chemical phenotype. Self-complementary replication in the pool occurs during a minority (here ≈ 35 %) of synthetic episodes that exploit coincidental overlaps between 4, 5 or 6 spikes of arbitrarily arriving substrates. Such uniquely productive synthetic episodes, in which near-ideal reaction sequences recur at random, account for most AB oligonucleotide synthesis, and therefore underlie the emergence of net replication under realistic primordial conditions. Because overlapping substrate spikes are unexpectedly frequent, and in addition, complex spike sequences appear disproportionately, a sporadically fed pool can host unexpectedly complex syntheses. Thus, primordial substrate fluctuations are not necessarily a barrier to Darwinism, but instead can facilitate early evolution.     

Simplifying principles for chemical and enzyme reaction kinetics

Tihonov's Theorems for systems of first-order ordinary differential equations containing small parameters in the derivatives, which form the mathematical foundation of the steady-state approximation, are restated. A general procedure for simplifying chemical and enzyme reaction kinetics, based on the difference of characteristic time scales, is presented. Korzuhin's Theorem. which makes it possible to approximate any kinetic system by a closed chemical system, is also reported. The notions and theorems are illustrated with examples of Michaelis-Menten enzyme kinetics and of a simple autocatalytic system. Another example illustrates how the differences in the rate constants of different elementary reactions may be exploited to simplify reaction kinetics by using Tihonov's Theorem. All necessary mathematical notions are explained in the appendices. The most simple formulation of Tihonov's 1st Theorem ‘for beginners’ is also given.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

Mutagenic activity and specificity of N-nitrosomethylaniline and N-nitrosodiphenylamine in Salmonella

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.     

Derivatives of side-chain hydroxylated nitrosamines direct acting mutagens in Salmonella typhimurium.

Methyl-(beta-tosyloxyethyl)nitrosamine and 3-methyl-4,5-dihydro-1,2,3-oxadiazolium tosylate are potent direct acting mutagens in the Ames assay, as is N-nitrosoprolinyl tosylate. These compounds are derived from beta-hydroxylated nitrosamines. The closely related methyl-(gamma-tosyloxypropyl)nitrosamine is not mutagenic without activation. These data are consistent with the chemical behavior of these substances, which suggest that suitable derivatives of beta-hydroxylated nitrosamines, such as O-sulfates, may be direct-acting biological alkylating agents.     

Calculation of steady-state rate equations and the fluxes between substrates and products in enzyme reactions.

1. Two methods are described for deriving the steady-state velocity of an enzyme reaction from a consideration of fluxes between enzyme intermediates. The equivalent-reaction technique, in which enzyme intermediates are systematically eliminated and replaced by equivalent reactions, appears the most generally useful. The methods are applicable to all enzyme mechanisms, including three-substrate and random Bi Bi Ping Pong mechanisms. Solutions are obtained in algebraic form and these are presented for the common random Bi Bi mechanisms. The steady-state quantities of the enzyme intermediates may also be calculated. Additional steps may be introduced into enzyme mechanisms for which the steady-state velocity equation is already known. 2. The calculation of fluxes between substrates and products in three-substrate and random Bi Bi Ping Pong mechanisms is described. 3. It is concluded that the new methods may offer advantages in ease of calculation and in the analysis of the effects of individual steps on the overall reaction. The methods are used to show that an ordered addition of two substrates to an enzyme which is activated by another ligand will not necessarily give hyperbolic steady-state-velocity kinetics or the flux ratios characteristic of an ordered addition, if the dissociation of the ligand from the enzyme is random.     

Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin)

The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-[4-azidobenzoyl]-N'-[(3-O-digoxigeninyl)methylcarbonyl)]-1 ,8-diamino- 3,6-dioxaoctane (photodigoxigenin), a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact.