Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.     

Correlation between Multi-Drug Resistance-Associated Membrane Transport in Clonal Cancer Cells and the Cell Cycle Phase

Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters, V(max) and K(M). Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater V(max) values, indicating a higher activity of transport. They also exhibited a greater V(max)/K(M) ratio, indicating a higher efficiency of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents.     

Shape and dynamics of thermoregulating honey bee clusters

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

An explicit solution for progress curve analysis in systems characterized by endogenous substrate production

The Lambert W function was used to explicitly relate substrate concentration S, to time t, and the kinetic parameters V (m), K (m), and R in the modified Michaelis-Menten equation that accounts for endogenous substrate production. The applicability of this explicit formulation for kinetic parameter estimation by progress curve analysis was demonstrated using a combination of synthetic and experimental substrate depletion data. Synthetic substrate depletion data were generated using S (0) values of 1, 2, and 3 μM and V (m), K (m), and R values of 1.0 μM h(-1), 1.0 μM, and 0.1 μM h(-1), respectively, and contained 5% normally distributed error. Experimental data were obtained from two previously published studies on hydrogen depletion in four experimental systems. In all instances, experimental data were well described by the explicit solution presented in this study. Differential equation solution and iterative S estimation are eliminated with the explicit solution approach, thereby simplifying progress curve analysis in systems characterized by endogenous substrate production.     

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.     

Kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm

The kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm during sorbitol oxidation were studied at pH 7.0, 7.5, 8.0, 9.0 and 10.0. It was found that the shape of kinetic curves for NADH accumulation depends on pH and substrate concentration. At pH 7.0, 7.5 and 8.0 the enzymatic reaction obeys the Michaelis-Menten kinetics with Km of 3.3 x 10(-3) M. 2.3 x 10(-3) M and 2.08 x 10(-3) M, respectively. At pH 9.0 and 10.0 the vovs [So] curves have an "intermediate plateau". The Hill plots for this reaction reveal two slopes that are dependent on substrate concentration. The nH values for sorbitol (up to 2 mM) are 1.0 and 1.16 at pH 9.0 and 10.0, respectively. With a further rise in the substrate concentration, the nH value increases up to 2.4 and 2.18 at pH 9.0 and 10.0, respectively. This is suggestive of the existence of a slowly dissociating enzymatic system of the Np in equilibrium P type (where P is the oligomeric and p the monomeric forms of the enzyme); N approximately greater than 2. The vovs NAD plots are S-shaped at all pH values studied. The data obtained are discussed in terms of regulatory effects of sorbitol and acidity on association-dissociation of sorbitol dehydrogenase from liver cell cytoplasm.     

A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K(m)=379μM and k(cat)=0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Thiorphan, tiopronin, and related analogs as substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase (PAM)

Peptidyglycine alpha-amidating monooxygenase is a copper- and zinc-dependent, bifunctional enzyme that catalyzes the cleavage of glycine-extended peptides or N-acylglycines to the corresponding amides and glyoxylate. This reaction is a key step in the biosynthesis of bioactive alpha-amidated peptides and, perhaps, the primary fatty acids amides also. Two clinically useful N-acylglycines are thiorphan and tiopronin, each with a thiol moiety attached to the acyl group. We report here that thiorphan and tiopronin are substrates for PAM, exhibiting relatively low K(M,app) and V(MAX,app) values. The low V(MAX,app) values result, most likely, from a decrease in active PAM.2Cu(II) as the enzyme competes ineffectively with thiorphan and tiopronin for free copper.     

Effect of dietary fat saturation on acylcoenzyme A:-cholesterol acyltransferase activity of Ehrlich cell microsomes.

Ehrlich cells grown in mice fed coconut oil diets (highly saturated) contain about twice as much cholesteryl ester as those grown in mice fed sunflower oil diets (highly polyunsaturated). Acylcoenzyme A: cholesterol acyltransferase (ACAT) activity was 30-100% higher in microsomes prepared from the cells grown on coconut oil (M(c)) than in those prepared from the cells grown on sunflower oil (M(s)). Increased ACAT activity was noted in M(c) with either [1-(14)C]palmitoyl CoA or [1,2-(3)H]cholesterol as the labeled substrate. This occurred at all acyl CoA concentrations tested and, in the [1,2-(3)H]cholesterol assay, with palmitoyl, oleoyl, or linoleoyl CoA as the substrate. The pH optimum for ACAT activity was the same with M(c) and M(s), pH 7.0. ACAT activity obeyed Michaelis-Menten kinetics at palmitoyl CoA concentrations between 1 and 10 micro M. Substrate inhibition occurred at higher concentrations. Kinetic analysis with [1-(14)C]palmitoyl CoA as the substrate indicated that the apparent K(m) for M(c) was 33% smaller than for M(s). There was no difference, however, in apparent V(max) values. The cholesterol and phospholipid contents of M(c) and M(s) were similar, but their fatty acid compositions differed considerably. M(c) contained 2.7 times more monoenoic fatty acid and only half as much polyenoic fatty acid as M(s). Our results indicate that dietary modification of the microsomal fatty acid composition is associated with alterations in the activity of ACAT, an enzyme that is tightly bound to the microsomes. These changes in ACAT activity may be partly responsible for the differences in cholesteryl ester contents of Ehrlich cells grown in mice fed the coconut and sunflower oil diets.     

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.     

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.     

The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.     

Analysis of negative cooperativity for glutamate dehydrogenase

The empirical equation, which describes negative cooperativity in the enzyme kinetics, has been proposed. The equation is obtained from the Michaelis-Menten equation where the Michaelis constant is replaced by the effective Michaelis constant, which is a linear function of the v/Vmax ratio (v is the rate of the enzymatic reaction and Vmax is the limiting value of v at saturating concentrations of substrate). The equation allows the limiting values of the Michaelis constant at v/Vmax --> 0 and V/Vmax --> 1 to be estimated, K0 and Klim, respectively. The Klim/K0 ratio is considered as a quantitative characteristic of negative cooperativity. The applicability of the equation has been demonstrated for the kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme). The negative cooperativity for the functions of saturation of protein by ligand is also analyzed. The data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase are used as examples.     

Evaluation of the kinetics of the intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes measured by amperometric methods

Amperometric methods were used to study the kinetics of intracellular reduction of 2,6-dichlorophenolindophenol (DCIP) in normal and transformed hepatocytes with glucose and succinate as substrates. The curves showing the formation of DCIPred as a function of time were biphasic, the first part obeying the equation of a pseudo-first-order reaction, the final part corresponding to Michaelis-Menten kinetics. A statistical method was used to estimate pseudo-first-order rate constants k as well as Km and Vmax values. At saturating glucose concentrations k, Km and Vmax values were higher in normal compared to transformed cells. Decreasing glucose concentrations revealed lowered saturation concentrations in tumour cells compared to normal cells. With succinate as substrate for hepatocytes, k values were higher than with glucose, while Km and Vmax were about the same. Hepatoma cells did not metabolize succinate. K values could be attributed to intracellular dehydrogenase activities including cytosolic and mitochondrial processes. Differences in pseudo-first-order rate constants between normal and tumour cells may therefore represent characteristic alterations associated with transformation.     

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).