Activity and Kinetic Properties of Phosphotransacetylase from Intestinal Sulfate-reducing Bacteria

Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransa-cetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophys-ical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V 0), maximum amount of the product of reaction (P max), the reaction time (half saturation period , τ) and maximum velocity of the phosphotransace-tylase reaction (V max) were defined. Michaelis constants (K m) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacety-lase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.


INTRODUCTION
Dissimilatory sulfate-reducing bacteria reduce inorganic sulfate or other oxidized sulfur forms to sulfide (Barton & Hamilton, 2010).This bacteria are heterotrophs and therefore, require an organic carbon source.In the case of Desulfovibrio and Desulfomicrobium genera, this carbon source can be supplied by simple organic molecules such as lactate, pyruvate, and malate.These are subsequently oxidized to acetate with the concurrent reduction of sulfate to sulfide (Rowan et al., 2009;Kushkevych, 2012a).
The process of organic compounds oxidation is a complex and multistage that provides the bacterial cells with energy (Kushkevych, 2012b).The lactate is the most common substrate used by the species belonging to the sulfate-reducing bacteria (Barton & Hamilton, 2010).This compound is oxidized to acetate via pyruvate (Sadana, 1954;Kushkevych, 2012a).
In our previous researches, we have demonstrated that lactate was oxidized incompletely to acetate by the intestinal sulfate-reducing bacteria D. piger Vib-7 and Desulfomicrobium sp.Rod-9 (Kushkevych, 2013).Lactate oxidation to acetate occurs with the intermediate compounds formation: pyruvate, acetyl-CoA and atsetylfosfat (Kushkevych, 2012a).
One important step in this degradative pathway involves the transfer of an acetyl group from acetyl-S-CoA to orthophosphate to form acetyl-PO 4 .The acetyltransferase catalyzing this reaction is phosphotransacetylase (acetyl-S-CoA: orthophosphate acetyltransferase, EC 2.3.18)(Kushkevych, 2012a): In the presence of sulfate, lactate in human intestine contributes to the intensive bacteria growth and the accumulation of their final metabolism product, hydrogen sulfide, which is toxic, mutagenic and cancerogenic to epithelial intestinal cells (Pitcher & Cummings, 2003;Rowan et al., 2009;Gibson et al., 1991;Kushkevych, 2012a).The increased number of the sulfate-reducing bacteria and intensity of dissimilatory sulfate reduction in the gut can cause inflammatory bowel diseases of humans and animals (Cummings et al., 2003;Gibson et al., 1991;Loubinoux et al., 2000;Kushkevych, 2012b).
As far as it is aware, phosphotransacetylase from intestinal sulfate-reducing bacteria D. piger and Desulfomicrobium has never been well-characterized.In literature, there are some data on phosphotransacetylase in various organisms as well as in the sulfate-reducing bacteria isolated from environment (Goldman, 1958;Reichenbecher & Schink 1997;Robinson & Sagers, 1972;Sadana, 1954;Shimizu, et al., 1969).However, the data on activity and the kinetic properties of this enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has not been reported yet.
The aim of this work was to study phosphotransacetylase activity in cell-free extracts of intestinal sulfate-reducing bacteria D. piger Vib-7 and Desulfomicrobium sp.Rod-9 and to carry out the kinetic analysis of enzymatic reaction.
The aim was accomplished using microbiological, biochemical, biophysical methods, and statistical processing of the results; the obtained data were compared with those from the literature.

MATERIALS AND METHODS
Objects of the study were sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp.Rod-9 isolated from the healthy human large intestine and identified by the sequence analysis of the 16S rRNA gene (Kushkevych, 2013;Kushkevych et al., 2014).
Bacterial growth and cultivation.Bacteria were grown in a nutrition-modified Kravtsov-Sorokin's liquid medium (Kushkevych & Moroz, 2012).Before seeding bacteria in the medium, 0.05 ml/l of sterile solution of Na 2 S×9H 2 O (1%) was added.A sterile 10N solution of NaOH (0.9 ml/l) in the medium was used to provide the final pH 7.2.The medium was heated in boiling water for 30 min in order to obtain an oxygen-free medium, and then cooled to 30°C.The bacteria were grown for 72 hours at 37°C under anaerobic conditions.The tubes were brim-filled with medium and closed to provide anaerobic conditions.
Obtaining cell-free extracts.Cells were harvested at the beginning of the stationary phase, centrifuged and suspended in 100 ml of 50 mM Tris(hydroxymethyl) aminomethane (Tris)-hydrochloride, pH 7.0 (henceforth referred to as Tris buffer), containing 1 mM ethylenediaminetetraacetate (EDTA).A suspension of cells (150-200 mg/ml) was obtained and homogenized using the ultrasonic disintegrator at 22 kHz for 5 minutes at 0°C to obtain cell-free extracts.The homogenate was centrifuged for 20 min at 16 000 g to remove the cell debris.The pellet was then used as the sedimentary fraction, and the supernatant obtained was termed the soluble fraction.The supernatant fluid and a Tris buffer wash of the pellet were subjected to a second centrifugation at 16 000 g for 40 min (Robinson & Sagers, 1972).The soluble extract constituted by the supernatant was used as the source of the enzyme.A pure supernatant, containing the soluble fraction, was then used as a cell-free extract.Protein concentration in the cell-free extracts was determined by the Lowry method (Lowry et al., 1951).
Assays for phosphotransacetylase activity.The phosphotransacetylase activity was assayed by measuring acetyl-P arsenolyzed in the presence of CoA as described previously in paper (Shimizu et al., 1969).A reaction mixture, containing 6 µmoles of acetyl-P, 15.8 nmoles of CoA (5 Lipmann units), 5 µmoles of cysteine, 20 µmoles of Tris-HC1 (pH 8.0), 50 µmoles of potassium arsenate (pH 8.0) and the enzyme in a final volume of 1 ml was incubated at 25°C for 12 min.The enzyme was diluted 10-250 times with 50 mM Tris-HC1 (pH 8.0) before incubation, and 50 µl of the solution were added to the mixture.One unit of phosphotransacetylase is defined as the amount of the enzyme which catalyzes the decomposition of µmole of acetyl-P under the specified conditions.Specific enzyme activity was expressed as U × mg -1 protein.The specific activity of the studied enzyme in the cell-free extracts of both bacterial strains under the effect of different temperature (20, 25, 30, 35, 40, 45°C) and pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0) in the incubation medium was measured.
Kinetic analysis.Kinetic analysis of the enzyme reaction was performed in a standard incubation medium (as it was described above) with modified physical and chemical characteristics of the respective parameters (the incubation time, substrate concentration, temperature and pH).The kinetic parameters characterizing the phosphotransacetylase reaction are the initial (instantaneous) reaction velocity (V 0 ), maximum velocity of the reaction (V max ), maximum amount of the reaction product (P max ) and characteristic reaction time (time half saturation) τ were determined.The amount of the reaction product was calculated stoichiometrically.The kinetic parameters characterizing phosphotransacetylase reactions such as Michaelis constant (K m ) and maximum reaction velocity of substrate decomposition were determined by Lineweaver-Burk plot (Keleti, 1988).For analysis of the substrate kinetic mechanism of phosphotransacetylase, initial velocities were measured under standard assay conditions with different substrate concentrations.The resulting data were also analyzed by global curve fitting in SigmaPlot (Systat Software, Inc.) to model the kinetic data for rapid equilibrium rate equations describing ordered sequential, ), and random sequential, V=(V- ), kinetic mechanisms, where V is the initial velocity, V max is the maximum velocity, K A and K B are the K m values for substrates A and B, respectively, and α is the interaction factor if the binding of one substrate changes the dissociation constant for the other (Segal, 1975).
Statistical analysis.Kinetic and statistical calculations of the results were carried out using the software MS Office and Origin computer programs.The research results were treated by the methods of variation statistics using Student's t-test.The equation of the straight line that the best approximates the experimental data was calculated by the method of least squares.The absolute value of the correlation coefficient r was from 0.90 to 0.98.The significance of the calculated parameters of line was tested by the Fisher's F-test.The accurate approximation was when P ≤ 0.05 (Bailey, 1995).

RESULTS AND DISCUSSION
Specific activity of phosphotransacetylase, an important enzyme in the process of organic compounds oxidation in sulfate-reducing bacteria, was measured in different fractions obtained from D. piger Vib-7 and Desulfomicrobium sp.Rod-9 cells (Table 1).Results of our study showed that the highest specific activity of the enzyme was detected in cell-free extracts (1.19 ± 0.122 and 0.37 ± 0.041 U × mg -1 protein for D. piger Vib-7 and Desulfomicrobium sp.Rod-9, respectively).The slightly lower values of activity of phosphotransacetylase were determined in the soluble fraction compared to the cell-free extracts.Its values designated 0.87 ± 0.091 U × mg -1 protein for D. piger Vib-7 and 0.32 ± 0.036 U × mg -1 protein for Desulfomicrobium sp.Rod-9.The enzyme activity in sedimentary fraction was not observed.
The effect of temperature and pH of the reaction mixture on phosphotransacetylase activity in the cellfree extracts of the sulfate-reducing bacteria was studied (Fig. 1).The maximum specific activity for both bacterial strains was determined at 30…35ºC.The highest enzyme activity of phosphotransacetylase for D. piger Vib-7 and Desulfomicrobium sp.Rod-9 was measured at pH 7.5…8.5.The enzyme activity exhibited typical bell-shaped curves as a function of temperature and pH.
Thus, temperature and pH optimum of this enzyme was 30…35ºC and pH 7.5…8.5,respectively.An increase or decrease in temperature and pH led to a decrease of the activity of studied enzyme in the cell-free bacterial extracts of the sulfate-reducing bacteria.
To study the characteristics and mechanism of phosphotransacetylase reaction, the initial (instantaneous) reaction velocity (V 0 ), maximum velocity of the reaction (V max ), maximum amount of reaction product (P max ) and reaction time (τ) were defined.Dynamics of reaction product accumulation was studied for investigation of the kinetic parameters of phosphotransacetylase (Fig. 2).
Experimental data showed that the kinetic curves of phosphotransacetylase activity have tendency to saturation (Fig. 2A).Analysis of the results allows to reach the conclusion that the kinetics of phosphotransacetylase activity in the sulfate-reducing bacteria was consistent to the zero-order reaction in the range of 0-3 min (the graph of the dependence of product formation on the incubation time was almost linear in this interval of time).Therefore, the duration of the incubation of bacterial cells extracts was 5 min in subsequent experiments.
Amount of product of phosphotransacetylase reaction in the D. piger Vib-7 was higher (15.43 ± 1.61 µmol × mg -1 protein) compared to the Desulfomicrobium sp.Rod-9 (4.56 ± 0.47 µmol × mg -1 protein) in the entire range of time factor.The basic kinetic properties of the reaction in the sulfate-reducing bacteria were calculated by linearization of the data in the {P/t; P} coordinates (Fig. 2B, Table 2).
The kinetic parameters of phosphotransacetylase from both D. piger Vib-7 and Desulfomicrobium sp.Rod-9 were significantly different.Values of initial (instantaneous) reaction velocity (V 0 ) for the enzyme was calculated by the maximal amount of the product reaction (P max ).As shown in Table 2, V 0 for phosphotransacetylase reaction was slightly higher (5.68 ± 0.58 µmol × min -1 × mg -1 protein) in D. piger Vib-7 compared to Desulfomicrobium sp.Rod-9 (2.14 ± 0.23 µmol×min -1 ×mg -1 protein).In this case, the values of the reaction time (τ) were more similar for the studied enzyme in both D. piger Vib-7 and Desulfomicrobium sp.Rod-9 strains.Based on these data, there is an assumption that the D. piger Vib-7 can consume lactate ion much faster in their cells than a Desulfomicrobium sp.Rod-9.Moreover, this hypothetical assumption can be also confirmed by obtained data on maximal velocities of accumulation of the final reaction products, where V max for enzyme reaction in D. piger Vib-7 were also more intensively compared to Desulfomicrobium sp.Rod-9 (Table 3).
The kinetic analysis of phosphotransacetylase activity dependence on concentration of substrate (acetyl-CoA) was carried out.The increasing of acetyl-CoA concentrations from 0.5 to 5.0 mM caused a monotonic rise of the studied enzyme activity and the activity was maintained on unchanged level (plateau) under substrate concentrations over 3.0 mM.(Fig. 2C).Clearly, the enzyme was saturated with substrate and the higher concentrations (3.0-5.0 mM acetyl-CoA) did not affect its activity, so the activity was maintained on unchanged (plateau) level.
Curves of the dependence {1/V; 1/[S]} were distinguished by the tangent slope and intersect the vertical axis in one point (Fig. 2D).The basic kinetic parameters  of phosphotransacetylase activity in D. piger and Desulfomicrobium sp.Rod-9 were identified by linearization of the data in the Lineweaver-Burk plot (Table 3).Calculation of the kinetic parameters of enzyme activity indicates that the maximum velocities (V max ) of acetyl-CoA in the D. piger Vib-7 and Desulfomicrobium sp.Rod-9 were significantly different from each other (P < 0.001).The values of K m were also quite different for acetyl-CoA (3.36 ± 0.35, 5.97 ± 0.62 mM) in both D. piger Vib-7 and Desulfomicrobium sp.Rod-9 strains, respectively.
The described results of the phosphotransacetylase activity and the kinetic properties of the enzyme in cellfree extracts of D. piger Vib-7 and Desulfomicrobium sp.Rod-9 strains are new and have never reported in the literature before.These obtained studies were differed significantly from previously described by Sadana (1954).A soluble enzyme system from Desulfovibrio desufuricans which catalyses the conversion of two moles of pyruvate to one mole of acetyl phosphate, one mole of ethyl alcohol, and two moles of CO 2 was described.The system required inorganic phosfate for pyruvate dissimilation.Pyrophosphate and arsenate could replace inorganic phosphate.The reaction was most rapid at pH 6.4.The optimum phosphate concentration was 12 M.The requirement of phosphate for the metabolism of pyruvate by the bacterial extract suggests the formation of acetyl coensyme A as an intermediate which is converted to acetyl phosphate and coenzyme A in the presence of inorganic phosphate by transacetylase.The effect is not due to hydrolysis of pyrophosphate to inorganic phosphate since the extracts show no appreciable pyrophosphatase activity (Sadana, 1954).
The phosphotransacetylase activity was also studied in crude extracts of Escherichia coli K-12 by Goldman in 1958.A little later, in 1969, Shimizu et al. have obtained and purified the phosphotransacetylase from crude extracts of Escherichia coli B with the use of ammonium  sulfate a stabilizer.The purified enzyme was homogeneous in ultracentrifugal analysis and molecular weight was tentatively estimated from the s value.K m values of this enzyme were 3×10 -3 and 4×10 -3 M for acetyl phosphate and 3.2×10 -4 M for CoA (Shimizu et al., 1969).The values of calculated K m of acetyl-CoA for phosphotransacetylase reaction in the cell-free extracts of D. piger Vib-7 and Desulfomicrobium sp.Rod-9 were slightly different to those previously described by Shimizu et al. (1969) Robinson andSagers (1972) have measured the activity of phosphotransacetylase from Clostridium acidiurici.Authors showed that the phosphotransacetylase has two properties not observed for this enzyme in other bacteria: it required a divalent metal for activity, and it was not subject to uncoupling by arsenate.The enzyme was obtained in highly purified form, with a specific activity 500-fold higher than crude extracts (Robinson & Sagers, 1972).
The described data were also different from results described by Reichenbecher and Schink (1997) for phosphotransacetylase activity from the Desulfovibrio inopinatus.Authors demonstrated that the activity was 44 mU (mg protein) -1 in extracts of ethanol-grown cells, while it was below 3 mU (mg protein) -1 after growth with hydroxyhydroquinone or lactate (Reichenbecher and Schink,1997).
Lawrence and co-authors ( 2006) have reported structural and functional studies suggesting a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila.Two crystal structures of phosphotransacetylase from the methanogenic archaeon M. thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft.Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1.The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate.Authors have proposed the mechanism of the reaction catalyzed by phosphotransacetylase from M. thermophila (Lawrence et al., 2006): Perhaps, the proposed mechanism for phosphotransacetylase from the methanogenic archaeon M. thermophila and the description given by Lawrence and co-authors (2006) may be similar to the studied enzymes from D. piger Vib-7 and Desulfomicrobium sp.Rod-9.However, to confirm this hypothetical assumption, the enzyme should be purified from cell-free extracts to study its structure and properties in detail.

CONCLUSIONS
The phosphotransacetylase, an important enzyme in process of dissimilatory sulfate reduction and lactate oxidation in sulfate-reducing bacteria, carries out the central step in oxidative decarboxylation of acetyl-CoA to acetyl-P.
The enzyme activity, V 0 and V max were significantly higher in the D. piger Vib-7 cells than Desulfomicrobium sp.Rod-9.However, Michaelis constants for acetyl-CoA were quite higher (5.97 ± 0.62 mM) in Desulfomicrobium sp.Rod-9 strain compared to D. piger Vib-7.The maximum enzyme activity for both strains was determined at +30…35ºC and at pH 7.5…8.5.The kinetic parameters of enzyme reaction are depended on the substrate concentration.
The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in the D. piger Vib-7 and Desulfomicrobium sp.Rod-9 intestinal strains, their production of acetate and hydrogen sulfide in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases.Data on the activity and kinetic properties of this enzyme in the strains can be useful to predict the velocity of the accumulation of the final products of metabolism of these bacteria, hydrogen sulfide and acetate, which are formed in the process of dissimilatory sulfate reduction.Assessing rate of formation of these dangerous products in the gut, we are able to predict their toxicity and occurrence of bowel diseases.

Figure 1 .
Figure 1.The effect of temperature (A) and pH (B) on the phosphotransacetylase activity in the cell-free extracts of the intestinal sulfate-reducing bacteria

Table 2 . Kinetic parameters of the phosphotransacetylase from intestinal sulfate-reducing bacteria
V 0 is initial (instantaneous) reaction velocity; P max is maximum amount (plateau) of the product of reaction; τ is the reaction time (half saturation period).Statistical significance of the values M ± m, n = 5; ** P < 0.01, *** P < 0.001, compared to the D. piger Vib-7 strain. Comment: