Interactions of monoamine oxidase inhibitors, N-acetylenic analogues of tryptamine, with rat liver microsomal cytochrome P450

Interactions between some novel and potent monoamine oxidase inhibitors (MAOIs), acetylenic analogues of tryptamine, and rat liver microsomal cytochrome P450 (P450) as evidenced by visible spectra analysis were analysed. Compounds with a secondary aliphatic amine moiety throughout induced type II difference spectra and exhibited the highest affinity for P450, whereas tertiary amines induced type I spectral changes and showed diminished affinity. P450 dependent aniline hydroxylase activity was inhibited by all compounds in an irreversible time-dependent manner. Only tertiary aliphatic amines constituted the substrate for P450-dependent N-demethylase activity, with comparable kinetic parameters. The N-demethylated metabolites were identified by thin-layer chromatography and mass-spectrometric analyses. These findings describe the role of P450-dependent microsomal mono-oxygenase systems in the metabolism of some MAOI acetylenic tryptamine derivatives and the possible hepatic contribution to adverse interactions between MAOIs, endobiotics and sympathomimetic compounds.     

Strain IMB-1, a novel bacterium for the removal of methyl bromide in fumigated agricultural soils

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class Proteobacteria and is closely related to members of the genus Rhizobium. The ability of strain IMB-1 to oxidize MeBr to CO2 is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.     

Chiral separation of amines by high-performance liquid chromatography after tagging with 4-(N,N-dimethylaminosulphonyl)-7-(2-chloroformylpyrrolidin-1-yl)- 2,1,3-benzoxadiazole

Chiral tagging reagents, 4-(N,N-dimethylaminosulphonyl)-7-(2-chloroformylpyrrolidin-1 -yl)-2,1,3- benzoxadiazole (R(+)-DBD-Pro-COCl and S(-)-DBD-Pro-COCl), react with mirror image enantiomers of amines to produce corresponding diastereomers in the presence of pyridine as a catalyst. The maximal excitation and emission wavelengths of the resulting diastereomers were ca. 450 nm and 560 nm, respectively. The diastereomers derived from some aliphatic amines were resolved by a reversed-phase chromatography with water-acetonitrile or normal-phase chromatography with n-hexane-ethyl acetate as the eluent. The reactivities of both enantiomers of DBD-Pro-COCl to chiral amines were almost comparable, whereas a slight difference of fluorescence intensity was observed with S(-)-DBD-Pro-COCl. When (S-)-DBD-Pro-COCl was used as the derivatization reagent, amines corresponding to S-configuration were eluted faster than R-configuration. The opposite elution order was obtained with the use of R(+)-DBD-Pro-COCl, instead of S(-)-DBD-Pro-COCl. The Rs values obtained from 1-cyclohexylethylamine (CEA) having aliphatic ring structure was larger than those of amines (1-(1-naphthyl)ethylamine (NEA) and 1-phenylethylamine (PEA)) having aromatic ring structures.     

Direct accessory optic projections to the vestibulo-cerebellum: a possible channel for oculomotor control systems

The nucleus of the basal optic root (nBOR) receives direct retinal projections in all classes of vertebrates. This nucleus is also known as the medial terminal nucleus of the accessory optic tract, or as the nucleus extomamillaris. Following a series of HRP injections into the uvula and flocculonodular lobe of pigeons, both large and small cells of the nBOR were labelled bilaterally with the marker. No significant transport of HRP to nBOR was observed following injections of more rostral folia of the posterior or anterior lobes of the cerebellum. In view of the prominence of the tract from retina to nBOR and the presence of a monosynaptic pathway from nBOR directly to the vestibulo-cerebellum, we suggest this bisynaptic, "lemniscal", retino-cerebellar channel may be the substrate by which visual stimuli directly trigger oculomotor responses mediated by the vestibulo-cerebellum.     

Accumulation of aliphatic amines in gastric juice of acute renal failure patients. Possible cause of hypergastrinemia associated with uremia

In this study we analyzed by gas chromatographic headspace analysis the composition and concentration of gastrin-stimulatory volatile aliphatic amines in the gastric juice of healthy subjects and acute renal failure patients. We demonstrated that although these aliphatic amines are present in the gastric juice of normal subjects in trace amounts, they accumulate in the gastric juice of uremic subjects. This 30-40-fold elevation in gastric juice amine concentration agreed favorably with the 40-50-fold augmentation in serum gastrin levels in acute renal failure, with a significant association (r = 0.87) existing between these two parameters. It was also determined that a 2-hr hemodialysis procedure resulted in a modest nonparallel decline in both gastric amine and serum gastrin levels. These results support the hypothesis that the accumulation of volatile aliphatic amines in the gastric juice of uremic individuals may induce an activation of the antral G cells, resulting in hypergastrinemia.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2

Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H2O2 with concurrent consumption of O2. Both adrenochrome formation and O2 consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H2O2. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O2- in the oxidation. Although SOD-mediated H2O2 formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O2- takes part in a vital reaction to form an intermediate for adrenochrome formation and O2 consumption. Generation of O2- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H2O2, both adrenochrome formation and O2 consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn2+ or spin-trap, alpha-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O2 consumption. The free radicals thus favor reduction of O2 rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe3+/Fe2+ shuttle consuming O2, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O2 to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN-, SCN-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O2- and H2O2. Once H2O2 is generated, the peroxidative cycle continues with the consumption of O2, through the intermediate formation of O2- and H2O2 which play an obligatory role in subsequent cycles of peroxidation.     

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.     

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity

BACKGROUND: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis. RESULTS: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. CONCLUSIONS: The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.     

Carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase

The carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) was examined using a recombinant enzyme. Kinetic parameters were obtained for 48 carbonyl group-containing substrates, including aromatic aldehydes, aromatic ketones, cycloketones, quinones, aliphatic aldehydes and aliphatic ketones. 20 beta-HSD showed a high affinity towards quinones, such as 9,10-phenanthrenequinone, alpha-naphthoquinone and menadione (Km values of 4, 2 and 5 microM, respectively), and the substrate utilization efficiency (Vmax/Km) of the enzyme against these quinones was very high. Cyclohexanone and 2-methylcyclohexanone were also reduced with a high Vmax/Km value, but not cyclopentanone or 2-methylcyclopentanone. Various aromatic aldehydes and ketones including benzaldehyde- and acetophenone-derivatives were reduced by 20 beta-HSD. Especially, 4-nitrobenzaldehyde and 4-nitroacetophenone were reduced with high Vmax/Km values in the related compounds. The enzyme also reduced the pyridine-derivatives, 2-, 3-, and 4-benzoylpyridine, with the Vmax/Km value for 2-benzoylpyridine being the highest. 20 beta-HSD reduced aliphatic aldehydes and aliphatic ketones, but was more effective on the former. The correlation between the structure of carbonyl compounds and their substrate Vmax/Km is discussed.     

Biological and toxicological effects of solvent extraction chemicals: Range finding acute toxicity in the rainbow trout (Salmo gairdnerii Rich.) and in the rat (Rattus norwegicus L.)

The acute toxicity of twelve solvent extraction chemicals was studied in the albino rat after intraperitoneal injection of the chemicals, and in the rainbow trout (Salmo gairdnerii Rich.) after exposure to the chemicals in the aquaria water. The tested chemicals included the following technical products: one primary aliphatic amine (Primene JM-T), two secondary aliphatic amines (Amberlite LA-1 and Adogen 283), two tertiary aliphatic amines (Adogen 383 and Alamine 336), one quaternary amine (Aliquat 336), two organophosphorus compounds (TBP, tributyl phosphate and HDEHP, di-(2-ethyl)-hexylphosphoric acid), one synthetic carboxylic acid (Versatic 10), one naphthalene-sulphonic acid (NA-SUL AS-50), one substituted oxime (LIX 64N) and one aliphatic alcohol (2-ethylhexanol). The intraperitoneal 96 hours LD 50 in the rat for the tested chemicals varied between less than 50 mg/kg b.w. to more than 5000 mg/kg b.w. Ninety six hours LC 50 in the rainbow trout was not possible to determine for three of the twelve tested chemicals due to their low water solubility. When determined, the LC 50 at 15°C was in the range 0.11–56 mg/l based on the amounts added.The present study discusses the acute toxicity of the chemicals mentioned above, gross pathological changes in the rat and behavioural changes in the fish.     

Cationic liposomes in gene delivery

Cationic liposomes have been extensively explored as gene delivery vector for several reasons. It is because disadvantages of viral vectors include risk of replication, possible immunogenicity, and the difficulty of obtaining a large quantity of viral vectors. Currently, a variety of cationic components for liposome formulations have been developed. The components are broadly divided into two classes based on the chemical structure of hydrophobic moieties: long aliphatic (saturated or unsaturated) hydrocarbons and cholesterol ring. A variety of hydrophilic moieties also include tertiary amines, ammonium salt and spermine. The role of liposomes is to condense DNA to form complexes with high affinity to cell surfaces where possible fusion or destabilization of the membrane and/or endocytosis are involved. However, at present, little structure-activity relationships are known. Some vectors are on clinical trials approved by NIH.     

Carboxylation of epoxides to beta-keto acids in cell extracts of Xanthobacter strain Py2

A novel enzymatic reaction involved in the metabolism of aliphatic epoxides by Xanthobacter strain Py2 is described. Cell extracts catalyzed the CO2-dependent carboxylation of propylene oxide (epoxypropane) to form acetoacetate and beta-hydroxybutyrate. The time courses of acetoacetate and beta-hydroxybutyrate formaton indicate that acetoacetate is the primary product of propylene oxide carboxylation and that beta-hydroxybutyrate is a secondary product formed by the reduction of acetoacetate. Analogous C5 carboxylation products were identified with 1,2-epoxybutane as the substrate. In the absence of CO2, propylene oxide and 1,2-epoxybutane were isomerized to form acetone and methyl ethyl ketone, respectively, as dead-end products. The carboxylation of short-chain epoxides to beta-keto acids is proposed to serve as the physiological reaction for the metabolism of aliphatic epoxides in Xanthobacter strain Py2.     

Intravenous immunoglobulin in oncology nursing practice

Phenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehydrogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different. We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase. The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme. In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively. These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.     

Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of manganese

A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures of Bjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (Mr = 43,000, pI 3.88, and epsilon407 nm = 123 mM-1 cm-1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (low Km of 51 microM and turnover number of 59 s-1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6-dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- and p-anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (Km = 116 microM) and its turnover number (2.8 s-1) are comparable to those of lignin peroxidase (LiP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mM severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor.     

A novel white laccase from Pleurotus ostreatus

Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a "white" laccase.     

The substrate specificity of cytochrome P450cam

The catalytic turnover of xenobiotics by cytochrome P450cam results in both the formation of organic metabolites and the uncoupled production of H2O2, and H2O. Previous studies have shown that a receptor-constrained three-dimensional screening program (DOCK) can be used to identify potential ligands (ergo substrates) for the enzyme (De Voss, J. J.; Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1997, 119, 5489). A new set of 10 compounds has now been examined to further test the substrate specificity of P450cam and the ability of DOCK to identify substrates for this enzyme. The results expand the known specificity of P450cam and define limitations in the use of DOCK to predict its substrate specificity.     

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.     

Dietary modulation of human platelet phenolsulphotransferase activity

1. The mammalian phenolsulphotransferase enzymes are known to play a major role in both the detoxification and possibly the activation of pre-carcinogenic phenols and aromatic amines. 2. Vegetable cytosol preparations were tested in vitro for their ability to affect the sulphation of two reference compounds (rho-nitrophenol and dopamine, which are selective substrates for the phenol and monoamine forms of phenolsulphotransferase respectively), and to act as substrates for the enzymes in comparison with the same reference compounds. 3. The majority of cytosols greatly decreased (> 80%) the sulphation of either or both the reference compounds. This effect may have been due to either enzyme inhibition or substrate binding. 4. Whereas some of the cytosols were sulphated under the assay conditions, most were not. Additionally, it was found that a cytosol that decreased the sulphation of the two reference compounds was not necessarily poorly sulphated itself. 5. It is concluded that dietary factors have the potential to play a major role in modulating the sulphation detoxification pathway, and have wide ranging implications with regard to adverse drug reactions.     

Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase

Mutation to the conserved Glu399 or Lys192 caused the rate-limiting step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurley, T. D., and Weiner, H. (1997) J. Biol. Chem. 272, 18817-18822). Here we further investigated the role of these two NAD+-ribose-binding residues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase activity when compared with the native enzyme. No pre-steady state burst of NADH formation was found with the E399Q/K and K192Q enzymes when propionaldehyde was used as the substrate; furthermore, each mutant oxidized chloroacetaldehyde slower than propionaldehyde, and a primary isotope effect was observed for each mutant when [2H]acetaldehyde was used as a substrate. However, no isotope effect was observed for each mutant when alpha-[2H]benzaldehyde was the substrate. A pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants. The rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates. The K192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.