Citrate synthase from Crithidia fasciculata: inhibition by adenine nucleotides and suramin

Citrate synthase (EC 4.1.3.7) was purified to electrophoretic homogeneity from Crithidia fasciculata ATCC 11745. 2. The purified enzyme had an optimal pH of 8.0-8.5, apparent Km values for acetyl-CoA and oxaloacetate of 5.5 and 3.5 microM, respectively, and was not activated by NH4Cl or KCl, nor inhibited by NADH or alpha-oxoglutarate. 3. Adenine nucleotides inhibited the enzyme, ATP being the most effective. The inhibition was strictly competitive towards acetyl-CoA and of the mixed type with respect to oxaloacetate. 4. The trypanocidal drug suramin inhibited both the C. fasciculata and the pig liver citrate synthases, being strictly competitive with respect to oxaloacetate, and non-competitive towards acetyl-CoA. The competitive inhibition with respect to the divalent anion oxaloacetate might be due to the strongly anionic nature of suramin, which has six sulfonic groups in its molecule.     

Acetyl-C0A hydrolase; activity, regulation and physiological significance of the enzyme in brown adipose tissue from hamster

1. Acetate production in hamster brown adipose tissue is a consequence of the existence of an acetyl-CoA hydrolase. The enzyme is soluble and is localised to the mitochondrial matrix. 2. Acetyl-CoA hydrolase has an apparent Km for acetyl-CoA of 51 muM and a specific acitivyty at 30 degrees C of 870 nmol of acetate formed/min per mg 100 000 X g supernatant protein. 3. The enzyme is noncompetitively activated by ADP and inhibited by NADH and the effect of these nucleotides may well serve to regulate the enzyme activity in vivo. 4. A strong product inhibition by CoA is observed. The inhibition is of S-linear-I-hyperbolic noncompetitive nature. 5. The hydrolase has a q10 of 2.0, which represents a 7.3% change in the rat of acetate production per degrees C. The energy of activation is12 200 cal/mol (53905 J/mol). 6. The regulatory role of acetyl-CoA hydrolase for fatty acid oxidation in brown adipose tissue of the hamster (a hibernator) at low as well as at normal body temperature is discussed.     

The metabolism of O-acyl-N-acylneuraminic acids. Biosynthesis of O-acylated sialic acids in bovine and equine submandibular glands

1. The enzymic synthesis of 4-O-acetylneuraminic acid, 4-O-acetyl-N-glycolyneuraminic acid, 4-O-glycolyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid and 9-O-acetyl-N-glycolyneuraminic acid is shown using radioactive precursors with surviving slices, membrane fractions or particle-free homogenates from bovine and equine submandibular glands. 2. Acetyl-CoA: N-acetylneuraminate-9(or 7)-O-acetyltransferase activity was found in a microsome fraction and in the cytosol of bovine submandibular glands. The properties of the membrane-bound enzyme acting on endogenous, glycoprotein-bound N-acetyl- and N-glycolylneuraminic acids were compared with those of the soluble enzyme, O-acetylating exogenous, non-glycosidically bound N-acetyl- and N-glycolyneuraminic acids. 3. A rapid, radioactive assay for the membrane-bound enzyme activity is described. The enzyme activity shows an optimum at pH 7 and has a Km for acetyl-CoA of 0.1 mM. The enzyme is inhibited by p-chloromercuribenzoate and iodoacetate. Divalent cations, EDTA and glutathione have no influence on its activity while CoA proved to be a competitive inhibitor with a Ki of 0.56 mM. 4. The soluble enzyme activity, assayed using a radioactive procedure, shows Km values of 0.01 mM, 0.5 mM and 0.39 mM for acetyl-CoA, N-acetylneuraminic acid and N-glycolylneuraminic acid respectively. The general properties are similar to those found for the membrane-bound enzyme, except that membrane-bound activity is stable for longer on storage at 4 degrees C. 5. Acetyl-CoA, acyl-CoA and CoA concentrations of 33 nmol, 65 nmol and 106 nmol/g wet tissue respectively are found in fresh bovine submandibular glands. 6. The occurrence of the CMP-glycosides of N-acetylneuraminic acid, 9-O-acetyl-N-acetyl-neuraminic acid and N-glycolylneuraminic acid in bovine submandibular glands is demonstrated. 7. The results are discussed in relation to the general metabolism of acylneuraminic acids.     

Female urethral syndrome. A female prostatitis?

N-Acetyltransferase, which is suggested to be responsible for the production of N1-acetylspermidine in Leishmania amazonensis and to be involved in the process of inactivation and degradation of excessive polyamines, was partially purified and characterized. Among the substrates tested, sym-norspermidine, sym-norspermine, and 1,3-diaminopropane had the highest reaction rates, but the naturally occurring polyamines spermine and spermidine were also acetylated at considerable rates, whereas putrescine was a poor substrate. The Michaelis constants (Km values) for spermine and spermidine were 0.66 and 3.3 mM, respectively. The Km value for acetylcoenzyme A (acetyl-CoA) was determined to be 34 microM. CoA inhibited the reaction in a competitive manner; the inhibition constant was 5 microM. The enzyme showed an apparent relative molecular mass of 35,000.     

Inhibitory actions of emodin on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori, a gram-negative rod bacteria collected from peptic ulcer patients. The NAT activity was determined using a acetyl CoA recycling assay and HPLC. Cytosols or suspensions of H. pylori with and without selected concentrations of emodin co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there were decreased NAT activity associated with increased emodin in H. pylori cytosols. As 400 microns of emodin can obviously inhibit NAT activity both in vitro and in vivo (inhibition rate 90% and 93% for 2-aminofluorene and p-aminobenzoic acid in vitro, and 90% and 92%, respectively, for both substrate in vivo). For in vitro examination, the apparent values of Km and Vmax were 3.12 +/- 0.38 mM and 15.20 +/- 3.16 nmol/min/mg protein for 2-aminofluorene, and 0.56 +/- 0.12 mM and 0.74 +/- 0.09 nmol/min mg protein for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 2.40 +/- 0.32 mM and 10.62 +/- 0.04 nmol/min/mg protein for 2-aminofluorene, and 0.23 +/- 0.02 mM and 0.62 +/- 0.08 nmol/min/mg protein for p-aminobenzoic acid. For in vivo examination, the apparent Km and Vmax were 0.82 +/- 0.18 mM and 0.92 +/- 0.21 nmol/min/10 x 10(10) colony forming units (CFU) for 2-aminofluorene, and 0.78 +/- 0.14 mM and 0.52 +/- 0.06 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 0.50 +/- 0.08 mM and 0.62 +/- 0.22 nmol/min/ 10 x 10(10) (CFU) for 2-aminofluorene, and 0.52 +/- 0.21 mM and 0.26 +/- 0.04 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. This report is the first finding of emodin inhibition of arylamine N-acetyltransferase activity in a strain of H. pylori.     

Inhibitory actions of glycyrrhizic acid on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) as substrates were determined in Helicobacter pylori, collected from patients with peptic ulcers. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from H. pylori demonstrated that glycyrrhizic acid elicited dose-dependent bactericidal effect in H. pylori cultures, i.e.; the greater the concentration of glycyrrhizic acid, the greater the inhibition of growth of H. pylori. Cytosols or suspensions of H. pylori with and without selected concentrations of glycyrrhizic acid co-treatment showed different percentages of 2-AF and PABA acetylation. The data indicated that there was decreased NAT activity associated with increased glycyrrhizic acid in H. pylori cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of Km and Vmax were decreased after co-treated with 80 M glycyrrhizic acid. This report is the first demonstration of glycyrrhizic acid inhibition of arylamine NAT activity and glycyrrhizic acid inhibition of growth in the bacterium H. pylori.     

Mycobacterium smegmatis fatty acid synthetase. Polysaccharide stimulation of the rate-limiting step

An initial activity burst lasting 5 to 10 s is observed for both de novo synthesis with acetyl-CoA as primer and for elongation of palmitoyl-CoA catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis. After the initial burst, synthetase activity slows at least 6-fold to the steady state rate. The size of the initial burst is proportional to the amount of synthetase protein and corresponds to the synthesis of a small number C three to five) of C24 or C26 acyl chains per mol of enzyme. During the initial burst, C24, C26 acyl enzyme is formed and can be isolated by ammonium sulfate precipitation. On incubation with CoA, enzyme-bound acyl chains undergo transacylation to form the corresponding CoA derivatives. Diffusion of C24-CoA and C26-CoA from the enzyme is slow and rate-limiting for overall fatty acid synthesis. Mycobacterial polysaccharides markedly accelerate this rate-determining step but bovine serum albumin does not. This facilitation of product diffusion accounts for the large stimulation of de novo synthesis and of elongation of mycobacterial polysaccharide. It is also shown that the high apparent Km for acetyl-CoA (approximately 400 micrometer) in the steady state reflects the substrate concentration required to shift the product pattern in favor of shorter chain fatty acids (C16,C18). These conditions circumvent the slow, rate-limiting diffusion of C24-CoA and C26-CoA.     

Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium. The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.     

Mycophenolate mofetil: therapeutic applications in kidney transplantation and immune-mediated renal disease

The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems, i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ibuprofen decreases the apparent Km and Vmax of NAT enzyme from human colon tumour cells in both systems examined. This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.     

Ketone bodies and brain glutamate and GABA metabolism

The effects of ketone bodies on brain metabolism of glutamate and GABA were studied in three different systems: synaptosomes, cultured astrocytes and the whole animal. In synaptosomes the addition of either acetoacetate or 3-OH-butyrate was associated with diminished consumption of glutamate via transamination to aspartate and increased formation of labelled GABA from either L-[2H5-2,3,3,4, 4]glutamine or L-[15N]glutamine. There was no effect of ketone bodies on synaptosomal GABA transamination. An increase of total forebrain GABA and a diminution of aspartate was noted when mice were injected intraperitoneally with 3-OH-butyrate. In cultured astrocytes the addition of acetoacetate to the medium was associated with a significantly enhanced rate of citrate production and with a diminution in the rate of conversion of [15N]glutamate to [15N]aspartate. These data are consistent with the hypothesis that the metabolism of ketone bodies to acetyl-CoA results in a diminution of the pool of brain oxaloacetate, which is consumed in the citrate synthetase reaction (oxaloacetate + acetyl-CoA --> citrate). As less oxaloacetate is available to the aspartate aminotransferase reaction, thereby lowering the rate of glutamate transamination, more glutamate becomes accessible to the glutamate decarboxylase pathway, thereby favoring the synthesis of GABA.     

An assay of flavin-containing monooxygenase activity with benzydamine N-oxidation

An assay system of flavin-containing monooxygenase was developed by fluorometric determination of benzydamine (BZY) N-oxidation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by rat liver microsomes was similar to that by purified FMO. The Km and Vmax values for the formation of N-desmethylbenzydamine (Nor-BZY) by rat liver microsomes were about 50 times greater and 2000 times less, respectively, than those of BZY N-oxide. Nor-BZY was not formed upon incubation with purified enzyme. BZY N-oxidation activity was completely inhibited both in the absence of NADPH and by heat inactivation. The reaction was inhibited in the presence of 0.5 mM thiourea, but 2 mM SKF-525A did not affect BZY N-oxidation. Moreover, rabbit antibody raised against the rat enzyme inhibited BZY N-oxidation. These results are in accord with a simple, rapid, and sensitive assay for the enzyme.     

The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

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.     

Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer

A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.     

Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods

We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.     

Determination of monoamine oxidase and catechol-O-methyltransferase in human blood components: methodical aspects (author's transl)

Controversal findings are reported with respect to alternations in activity of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) in psychoses. Initially we determined the interindividual differences of some biochemical properties of the two enzymes in normal control subjects. Platelet rich plasma and lysate of red blood cells, respectively, were used for assay. Enzyme activity was referred to mg of protein or mg hemoglobin and number of platelets, respectively. Substrates used for COMT assay were: 3,4-dihydroxybenzaldehyde and 3,4-dihydroxybenzoic acid; for MAO determination: tyramine, tryptamine and phenylethylamine. Interindividual as well as intraindividual differences in the biochemical characteristics (apparent Km, Vmax, IC50, meta/para ratio of O-methylation in vitro) were remarkably low, the coefficient of variation was in the range of 30%.     

Monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

The acyl-CoA:acyl-sn-glycerol 3-phosphate acyltransferases located in the microsomal fraction of lactating bovine mammary tissue show a preference for palmityl-CoA particularly above the apparent Km values of the acyl acceptors. Using saturating levels of monopalmityl-sn-glycerol 3-phosphate, the order of acylation was palmityl- greater than myristyl- greater than oleyl- greater than stearyl- greater than linoleyl-CoA. Apparent Km values for monopalmityl- and mono-oleyl-sn-glycerol 3-phosphate with palmityl-CoA as donor were 16 and 13muM, respectively while the Km values for palmityl-CoA with these two acyl acceptors were 5 and 5.2muM, RESPECTIVELY. The apparent Vmax values for the palmityl acceptor and donor were 25 and 30 nmol/min/mg protein. Phosphatidic acid was the principal product. The inclusion of magnesium in the assay depressed activity while the addition of ethylenediaminetetraacetate doubled the rate of acylation.     

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.     

Simulation of the enzyme reaction mechanism of malate dehydrogenase

A hybrid numerical method, which employs molecular mechanics to describe the bulk of the solvent-protein matrix and a semiempirical quantum-mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride transfers associated with MDH and cofactor nicotinamide adenine dinucleotide (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with proton transfer preceding hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction, resulting in energetics similar to that found in aqueous solution. An energy decomposition analysis indicates that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH.     

Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases

The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast. The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect. Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM). 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs. 0.4 mM; apparent Vmax: 0.3 vs. 0.2 U/ microg protein). In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation. The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs. 0.04 microM (ethanol). The KI for ethanol in baker's yeast was 470 microM. In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain.