- Email: [email protected]
Kinetic characteristics of 1-en-dehydrogenation of 6α-methylhydrocortisone by cells of Arthrobacter globiformis 193
ELSEVIER
Kinetic characteristics of 1-en-dehydrogenation of 6
Moscow
and Physiology
Region,
of Microorganisms,
Russian Academy
of Sciences,
Russia
Biochemical
characteristics of 6a-methylhydrocortisone I-en-dehydrogenation by bacterial cells of Arthrobac193 have been studied. The reaction ,follows the kinetics of substrate inhibition: the inhibition reveals itself at the level of the respiratory chain. A mathematical model describing multiple substrate inhibition during the process of 60c-methylhydrocortisone l-en-dehydrogenation with whole cells was proposed. The solution of the constructed model agrees well with experimental data. There is little or no inhibitory effect at low substrate concentrations, and the reaction rate is determined by the enzyme-substrate interaction rather than the 0 1996 Elsevier Science Inc. respiratory chain activity. ter globiformis
Keywords: 3-ketosteroid-1Lendehydrogenase; Arthrobucter globiformis; 6a-methylhydrocortisone; microbiological transformation; substrate inhibition; mathematical model; kinetic parameters
Introduction Microbiological 1-en-dehydrogenation of steroids is known to be the key process in the synthesis of glucocorticoids. As a rule, the dehydro derivatives are physiologically more active compounds than their precursors. For example, 6a-methylprednisolone is known to have higher antiinflammatory, antiallergic, and other activities in comparison with 6o-methylhydrocortisone and exert fewer side effects.’ In spite of the great practical interest of this process, its biochemical and kinetic features have not yet been studied enough. As was shown for hydrocortisone l-en-dehydrogenation (prednisolone production) by Arthrobacter globiformis 193 cells2 this process involves the respiratory chain in line with 3-ketosteroid-1-en-dehydrogenase (EC 1.3.99.4). This enzyme is localized in the cytoplasmic membrane and transfers presumably the reducing equivalents to menaqui-
dehydrogenation;
none. The final acceptor of electrons and the second substrate of the reaction is oxygen. The electron transfer step to the respiratory chain is the limiting step for the process as a whole. The reaction follows MichaelisbMenten kinetics. Calculated parameters reflect electron transfer along the chain of carriers.3 Introduction of an additional methyl group into ring B of a steroid molecule may result not only in change in its physicochemical and therapeutic properties but also in the alteration of the l-en-dehydrogenation kinetics. The aim of this work is to study the characteristics of 6a-methylhydrocortisone l-en-dehydrogenation by whole bacterial cells of A. globiformis 193.
Materials and methods Strain
and cultivation
conditions
The bacterium A. globiformis 193 was provided by the culture collection of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino, Russia). Address reprint requests to Dr. Anna Yu Arinbasarova, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospekt Nauki 5, Pushchino, Moscow Region 142292, Russia Received 13 June 1995; revised 18 December 1995; accepted 10 January 1996
Enzyme and Microbial Technology 19:501-506, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Growth conditions and the cell biomass assay were performed as described previously.4 Cortisone acetate was used as an inducer of 3-ketosteroid-lLen_dehydrogenase. The inducer was added in the form of an ethanol solution simultaneously with the inoculum at a concentration of 100 kg ml-‘.
0141-0229/96/$15.00 PII SO141-0229(96)00061-O
Papers Steroid transformation 6a-Methylhydrocortisone transformation by A. glohiformis 193 cells was performed in 750-ml flasks containing 100 ml of 0.01 M Na phosphate buffer pH 7.2 on a shaker at 200 rpm and 28°C. The substrate was added to the reaction medium as a methanol solution. The final content of methanol did not exceed 5% (v/v). Preliminary results suggested that the methanol at concentrations up to 7% (v/v) does not affect the reaction kinetics. The cell concentration was 0.1-1.0 g 1-l depending on substrate concentration.
Steroid analysis The degree of 6a-methylhydrocortisone transformation into 6amethylprednisolone was determined by semiquantitative TLC on Silufol UV-254 plates or by quantitative spectrophotometry.3,’ It should be noted that a decrease in the substrate and accumulation of the product occurred stoichiometrically; the dynamics of the process was determined by product accumulation. Reaction
rate
The kinetic curves for 6a-methylhydrocortisone
transformation were obtained using weighted approximation of experimental data by cubic splines.5 The activity of cells (reaction rate) was found as the maximal first derivative along the curve. The reaction rate was expressed in units of specific activity (nmol min-’ mg-’ dry weight cells).
Oxygen consumption Oxygen consumption by cells was determined with a polarograph LP-7 (from the former Czechoslovakia) using a Clark-type platinum electrode covered with Teflon film.2 Oxygen concentration in the medium was assumed to be 250 FM. Tris phosphate buffer (50 mM) pH 7.0 was used as medium. The measurement temperature was 22-25°C. The final sample volume was 2 ml. The cell concentration in the cuvette was 1.0 g 1-l dry weight.
Results and discussion Kinetics of 6cx-methylhydrocortisone I-en-dehydrogenation Figure I shows the kinetic curves of 6a-methylhydrocortisone transformation by A. globifomzis 193 cells. As seen from the Figure, the kinetic curves of product accumulation indicate the elongation of the lag period as substrate concentration increases. Since a decrease in the substrate and accumulation of the product occurs stoichiometrically, the similar lag period is also observed on the curves of substrate consumption (not presented in the Figure). Figure 2 represents the reaction rate dependence on substrate concentration. The features of this dependence allow one to suggest the kinetics of substrate inhibition. Maximal reaction rate is observed when the introduced substrate concentration is about 1.6 mu. The sufficiently high degree of transformation (up to 99%) when substrate concentration ranged from 0.2663.192 mu and the features of kinetic curves of the reaction product accumulation allow one to exclude inhibition by the product. It should be noted that at high concentrations of 6amethylhydrocortisone exceeding the limit of solubility in water (lower than 0.266 mM>, it is difficult to determine the 502
Enzyme Microb. Technol.,
I
I
0
I
160
timeSO Figure 1 1-En-dehydrogenation of 6a-methylhydrocortisone by A. globiformiscells. Substrate concentrations were 0.266 mM (O), 0.532 mM (W), 1.064 mM (A), 1.33 mM (+ ), and 1.596 mM (v). Cell concentration was 0.5 g I-’ (dry wt). The results of spline approximation are shown by solid lines
actual concentration in transformation medium. We assume that the kinetics of substrate inhibition may be manifested more clearly with the correspondence of introduced and actual 6c+methylhydrocortisone concentrations in the transformation medium. During the study of 6c+methylhydrocortisone dehydrogenation by Arthrobacter simplex cells entrapped in karraginane geL6 the effect of substrate inhibition at substrate concentrations up to 4.522 mM was not observed. This fact does not correlate with our data at first sight. This discrepancy may be caused by different experimental conditions. In the research of Pinheiro,6 the transformation medium was supplied with menadione as an artificial electron acceptor. Menadione radically changes the kinetic mechanism of the reaction from one characteristic to the native cells having natural electron acceptors. Moreover, heated immobilized cells were used and the reaction was performed in the presence of methanol at a concentration up to 20% (v/v). Interconnection
between steroid l-en-dehydrogena-
tion and oxygen consumption In studies of hydrocortisone transformation,2.3 it was shown that microbiological dehydrogenation is accompanied by oxygen consumption and controlled by the activity of the respiratory chain. As it turns out, the transformation of the methyl derivative of hydrocortisone was also accompanied by oxygen consumption. It was interesting to investigate the interconnection between the two processes. Figure 3 presents the initial rate of oxygen consumption by bacterial cells depending on substrate concentration. The initial oxygen consumption rate grows as the 6a-methylhydrocortisone concentration increases to 1 mM. A further increase in steroid concentration results in inhibition of respiration. These data are in good agreement with the results demonstrating the change of the initial transformation rate. Notice that there is no stoichiometry between substrate conversion and oxygen consumption. This fact is probably due
1996, vol. 19, November
15
Arthrobacter V (nmole/min/mg
0
dehydrogenation
of cells, dry wt)
::::I::::I~:::I::::I:II:II/ 0 5 10
of 6 alpha-methylhydrocortisone:
25
0
20
6a-methylhydrocortisone
et al.
10
(a)
15
A. Y. Arinbasarova
25
1
2
Substrate concentration
3
4
(mM)
(mM)
l/V, min . (mg of cells, dry wt) / nmole
Figure 3 The effect of substrate concentratilon on initial rates of transformation (M) and oxygen consumption (A) by A. globiformis cells
(b)
0.25
0.20 -b 0.15 --
0.10 ;0.05 --
0
::::;::::;::::;::;, 0
1
2
3
l/mM
Figure 2 The influence of substrate concentration on reaction rate in direct (a) and double reciprocal (b) coordinates (experimental data and model solution)
to competition between 3-ketosteroid-l-endehydrogenase and endogenous substrate dehydrogenases for the respiratory chain. Figure 4 illustrates the results of the investigation of the action of Go-methylhydrocortisone on oxygen consumption by A. globiformis cells in detail. One can see that 6c+methylhydrocortisone at a concentration of 1.33 mu results in cell respiration at a twofold rate (curve 1). The subsequent addition of an oxidative phosphorylation uncoupler, carbonylcyanide-chlorophenylhydrasone (CCCP) results in a further increase in the oxygen consumption rate (curve 1); however, there is no increase in the dehydro derivative accumulation (Figure 5). Evidently, the increase in the oxygen consumption rate is due to acceleration in the oxidation of endogenous substrates. Figure 4 (curve 2) shows that 6a-methylhydrocortisone at a concentration of 2.13 mu does not stimulate respiration of cells. Moreover, it inhibits respiration caused by oxidation of endogenous substrates. It should be noted that addi-
tion of CCCP to reaction medium with substrate concentrations of 2.13 mM and higher does not result in acceleration of both oxygen consumption and steroid transformation (data not shown). Supposedly the inhibition effect of 6cr-methylhydrocortisone on the transformation process is caused by direct interaction with steroid dehydrogenase; however, this supposition does not explain the inhibition effect of steroid on the endogenous respiration of cells (Figure 4, curve 2). The absence of the CCCP accelerating effect on respiration with high steroid concentrations is also not cllear. Alternatively, the inhibition effect of steroid is due to its ability to slow down electron transfer along the respiratory chain. To verify this supposition, we also study the action of 6a-methylhydrocortisone on respiration of cells which grow without the inducer and do not have any steroid dehydrogenase activity. Data in Figure 4 show that even a low concentration of 6a-methylhydrocortisone (1.33 mu) inhibits endogenous respiration of cells (curve 4). The inhibition effect is manifested more clearly in the background of the stimulating action of CCCP (curve 5) or valinomycine as a protonophor of potassium ions (curve 6) on cell respiration. These data demonstrate directly that the inhibition effect of 6cY-methylhydrocortisone on respiration is not due to the interaction with steroid dehydrogenase but the hindering of electron transfer along the respiratory chain. It should be noted that hydrocortisone at concentrations equal to or greater than 1.33 mM reveal no inhibition effect on respiratory activity of both induced and uninduced cells (data not shown). The effect of phenazine methasulfate (PMS) and menadione on 6a-methylhydrocortisone transformation and oxygen consumption by cells grown in the presence of steroid dehydrogenase inducer was also studied. Figure 4 (curve 3) shows that while the addition of 6a-methylhydrocortisone at a concentration of 2.13 mu results in inhibition of respiration, the subsequent introduction of PMS is followed by a marked acceleration of both respiration and transformation
Enzyme Microb. Technol.,
1996, vol. 19, November
15
503
Papers Cells
63 conversion
(%)
1 42-
21-
o1
I
I
80
40 time (min)
0
Figure 5 I-En-dehydrogenation of Ga-methylhydrocortisone by A. globiformis cells in the presence of PMS ( +), menadione (A), and CCCP (W. The control is 0. The Ga-methylhydrocortsone concentration was 1.33 mM. The cell concentration was 0.7 g I--’ (dry wt); PMS and menadione were 0.1 mM; and CCCP was 5 FM.
Methylhydrocortisone, i -33 trdl
bgthylhydroccrtiscne. 1.33 mY
1 min
Figure 4 The effect of Go-methylhydrocortisone on oxygen consumption by A. globiformis cells. Concentration of cells was 1.0 g I-’ (dry wt); phenazine methasulfate (PMS) and menadione were 0.1 mM; CCCP was 5 PM; and valinomycin was 0.2 pM. Curves 1,2, and 3 correspond to the cells grown in the presence of inducer. Curves 4, 5, and 6 correspond to the cells grown without an inducer of 3-ketosteroid-I-en-dehydrogenase. Numbers on the curves indicate the respiration rate in nmoles O2 min-’ mg-’ of cells (dry wt)
of the substrate. The reaction rate increased 1.5-fold (Figure 5). Similar results were obtained with menadione. The stimulating effect of PMS and menadione on oxygen consumption and steroid transformation activity is probably caused by the fact that these artificial electron acceptors are capable of accepting the reducing equivalents from steroid dehydrogenase and transferring them directly to oxygen. In this case, the site of electron transfer inhibition by 6a-methylhydrocortisone is shunted. The above findings can also explain the lack of inhibition of 6e+methylhydrocortisone dehydrogenation by A. simplex cells in the presence of menadione.6 The absence of an acceleration in transformation after adding CCCP can be presumably considered due to a lack of control of this process by respiratory chain activity. As to the diffusion control, if the reaction rate is limited by substrate diffusion, we would observe a decrease in the lag period for product accumulation at 6c+methylhydrocortisone concentrations greater than 0.8 mu (Figure I) rather than an increase. Thus, the data show that inhibition of 6ol-methylhydrocortisone is observed on the respiratory chain probably at the level of menaquinone-cytochrome b. 504
Enzyme Microb. Technol.,
Mathematical
model
To simplify the mathematical description of the dependence of reaction rate on substrate concentration, we offer three suggestions. Firstly, the enzyme (E) can be considered a functional unit localized in a cytoplasmic membrane; it includes 3-ketosteroid-len-dehydrogenase and the respiratory chain (menaquinone and cytochromes a, b, and c). The latter regenerates an active form of the enzyme. Secondly, the above functional unit can interact with both one and several substrate molecules (S, 6a-methylhydrocortisone) forming ES and S,E complexes, respectively; the reaction rate may decrease in the latter case (multiple substrate inhibition). Lastly, the &ES complex cannot form the product P. The kinetic scheme of the reaction corresponding to the above suggestions is presented in the Figure 6. Then one can describe the reaction rate by the equation similar to Webb’s equation’:
ES
E
-
E+ P
\
//
Sn ES
\ S,E
R - + E
P + (n-1)s
Figure 6 Kinetic scheme of the reaction. E is a functional unit including 3-ketosteroid-I-en-dehydrogenase and the respiratory chain, S is the substrate (6a-methylhydrocortisone), and Pis the product (&-methylprednisolone). ES, S,E, and S,fS correspond to different types of E and S interactions
1996, vol. 19, November
15
Arthrobacter
v=v
dehydrogenation
ws+Pw, m”* 1 +
w, + w, + w,wfh
of 6 alpha-methylhydrocortisone:
(1)
where Ws = [S]/K,, W, = ([S]/yK&“, and [S] is the substrate concentration. The parameters of the model are presented in Table 1. The given values of the model parameters were determined by minimization of the function representing the sum of squares of the model solution deviations from experimental data. The simplex method and the quasi-Newton method implemented in the MINUIT system of programs’ have been used to solve the minimization problem. Figure 2 presents the model solution in direct and double inverse coordinates after data fitting. As can be seen from the Figure, the model describes the experimental data well enough, which confirms the above suppositions indirectly. Since the parameter p is not equal to zero, the reaction cannot be referred to any classical type of inhibition.7 The formation of the S,E complex could be due to binding of steroid on the cytoplasmic membrane and its modification which results in multiple substrate inhibition. Furthermore, the formation of microcrystals (or pseudocrystals) could also take place as shown for hydrocortisone earlier.9.‘o Parameter values obtained by data fitting may also explain the observed elongation of the lag period with an increase in the 6a-methylhydrocortisone concentration. Indeed, the higher 6cx-methylhydrocortisone concentration, the greater quantity of substrate molecules is bound nonspecifically with the cell membrane in a quasistationary state. The above dependence is essentially nonlinear (n % 1). As a result, the achievement of a quasistationary state characterized by the maximal rate of product formation requires a long period. It should be especially noted that the obtained values of parameters V,,,, and K, coincide with ones calculated by the linear segments of the diagram in double inverse coordinates (at a substrate concentration from 0.2661 S96 mM). It is due to the high value of parameter II and the actual lack of the inhibition effect of the substrate within stated limits.
Table 1 Parameter Vmax &I n a
Kinetic parameters value, dimension,
A. Y. Arinbasarova
et al.
The Vma,value
obtained for 6a-methylhydrocortisone (21.2 nmole min-’ mg-’ cells) is about 8 times lower than one obtained earlier for hydrocortisone (160 nmole min-’ mg-’ cells.’ This conforms with the previous supposition that the limiting stage of the process at low substrate concentrations (up to 1.6 mM) is an enzyme-substrate interaction rather than the respiratory chain activity. At the higher substrate concentrations, as mentioned above. substrate inhibition takes place at the level of the cytochrome system. As to the KM value obtained for 6ar-methylhydrocortisone, it is about 7 times higher than those obtained earlier for hydrocortisone. namely 90 yM. This fact perhaps reflects the decrease in efficiency of the ES complex formation as compared with that for hydrocortisone.
Conclusion Introduction of an additional methyl group into hydroconisone results in a change in the kinetics of microbiological dehydrogenation. The process as a whole follows substrate inhibition kinetics rather than Michaelis-Menten kinetics. The inhibition effect of 6cr-methylhydrocortisone is revealed at the level of electron transfer along the respiratory chain. The solution of the constructed model agrees with experimental data. The model parameter values after data fitting imply that multiple substrate inhibition takes place. At low substrate concentrations (up to 1.6 tnM), there is little or no inhibitory effect and the reaction follows Michaelis-Menten kinetics. The kinetic parameter values after data fitting show that it is the 6a-methylhydrocortisone interaction with 3-ketosteroid1-en-dehydrogenase that is the limiting stage for the process.
Acknowledgments The authors thank Dr. V. A. Andrjushina from the Center of Bioengineering in Russia for providing 6ol-methylhydrocortisone.
of the reaction
References
and meaning
21.2 nmole min-’ mg-’ of cells (dry wt); the maximal rate 594 PM; the dissociation constant for the ES complex 12.6; the apparent number of substrate molecules for the S,E complex formation 747; the factor characterizing the affinity change in the S,ES complex formation 0.193; the’iactor characterizing the change of rate of product formation by the S,E complex as compared to the ES complex; 4.0; the factor characterizing the change in the dissociation constant for the S,E complex as compared to the ES complex yKhrl = 2.38 mM; the dissociation constant for the .S,E complex
Spero, G. B., Thompsone, J. L., Magerlein, B. J., Hanze, A. R., Murray, H. C., Sebek, 0. K.. and Hogg. J. A. Adrenal hormones and related compounds. IV. 6-methyl steroids. .I. Am. Chem. Sot. 1956. 78, 6213-6214 Medentsev, A. G., Arinbasarova, A. Yu., Koshcheyenko, K. A., Akimenko, V. K., and Skryabin. G. K. Regulation of 3-ketosteroidI-en-dehydrogenase activity of Arthrobacter ,gfobiformis cells by a respiratory chain. J. Steroid Biochrm. 1985. 23, 365-368 Arinbasarova, A. Yu., Medentsev. A. G., Akimenko, V. K., and Koshcheyenko, K. A. Kinetic regularities of hydrocortisone dehydrogenation and oxygen uptake by free and adsorbed Arthrobacrer globiformis cells. Biochemistry (Moscow) 1984, 49, 92G927 Koshcheenko. K. A., Sukhodolskaya. G. V., Tyurin. V. S., and Skryabin, G. K. Physiological, biochemical, and morphological changes in immobilized cells during repeated periodical hydrocortisone transformations. Eur. .I. Appl. Microbial. Biotechnol. 198 I. 12. 161-169
Enzyme Microb. Technol.,
1996, vol. 19, November
15
505
Papers 5. 6.
7. 8.
506
Marchuk, G. I. Methods of Computational Muthemutics. Nauka, Moscow, 1980, 138-145 (in Russian) Pinheiro, H. M. Bioconversion of 6o-methylhydrocortisone by immobilized cells of Arthrobucter simpfe-x. MSc. thesis. Instituto Superior Tecnico, Lisboa, 1988. abstract Webb, J. L. Enzyme andMetabolic fnhibizors. Generaf Principles of Inhibition. Mir, Moscow, 1966, 70-73 (in Russian) James, F. and Roos, M. MINUIT-a system for function mini-
Enzyme Microb. Technol.,
9.
10.
1996, vol. 19, November
mization and analysis of the parameter errors and correlations. Comp. Phvs. Commun. 1975, 10,343-367 Kondo, E. and Masuo. E. Pseudo-crystallofermentation of steroid:A new process for preparing prednisolone by microorganisms. ./. Grrr. Appl. Microbial. 196 1, 7, 113-l 17 Chen, K.-C., Chang, C.-C., Chiu, C.-F., and Ling, A. C. Mathematical stimulation of pseudo-crystallofermentation of hydrocortisone by Arthrobacter simpler Biotechnol. Bioeng. 1985, 27, 253-259
15
Kinetic characteristics of 1-en-dehydrogenation of 6
Moscow
and Physiology
Region,
of Microorganisms,
Russian Academy
of Sciences,
Russia
Biochemical
characteristics of 6a-methylhydrocortisone I-en-dehydrogenation by bacterial cells of Arthrobac193 have been studied. The reaction ,follows the kinetics of substrate inhibition: the inhibition reveals itself at the level of the respiratory chain. A mathematical model describing multiple substrate inhibition during the process of 60c-methylhydrocortisone l-en-dehydrogenation with whole cells was proposed. The solution of the constructed model agrees well with experimental data. There is little or no inhibitory effect at low substrate concentrations, and the reaction rate is determined by the enzyme-substrate interaction rather than the 0 1996 Elsevier Science Inc. respiratory chain activity. ter globiformis
Keywords: 3-ketosteroid-1Lendehydrogenase; Arthrobucter globiformis; 6a-methylhydrocortisone; microbiological transformation; substrate inhibition; mathematical model; kinetic parameters
Introduction Microbiological 1-en-dehydrogenation of steroids is known to be the key process in the synthesis of glucocorticoids. As a rule, the dehydro derivatives are physiologically more active compounds than their precursors. For example, 6a-methylprednisolone is known to have higher antiinflammatory, antiallergic, and other activities in comparison with 6o-methylhydrocortisone and exert fewer side effects.’ In spite of the great practical interest of this process, its biochemical and kinetic features have not yet been studied enough. As was shown for hydrocortisone l-en-dehydrogenation (prednisolone production) by Arthrobacter globiformis 193 cells2 this process involves the respiratory chain in line with 3-ketosteroid-1-en-dehydrogenase (EC 1.3.99.4). This enzyme is localized in the cytoplasmic membrane and transfers presumably the reducing equivalents to menaqui-
dehydrogenation;
none. The final acceptor of electrons and the second substrate of the reaction is oxygen. The electron transfer step to the respiratory chain is the limiting step for the process as a whole. The reaction follows MichaelisbMenten kinetics. Calculated parameters reflect electron transfer along the chain of carriers.3 Introduction of an additional methyl group into ring B of a steroid molecule may result not only in change in its physicochemical and therapeutic properties but also in the alteration of the l-en-dehydrogenation kinetics. The aim of this work is to study the characteristics of 6a-methylhydrocortisone l-en-dehydrogenation by whole bacterial cells of A. globiformis 193.
Materials and methods Strain
and cultivation
conditions
The bacterium A. globiformis 193 was provided by the culture collection of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino, Russia). Address reprint requests to Dr. Anna Yu Arinbasarova, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospekt Nauki 5, Pushchino, Moscow Region 142292, Russia Received 13 June 1995; revised 18 December 1995; accepted 10 January 1996
Enzyme and Microbial Technology 19:501-506, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Growth conditions and the cell biomass assay were performed as described previously.4 Cortisone acetate was used as an inducer of 3-ketosteroid-lLen_dehydrogenase. The inducer was added in the form of an ethanol solution simultaneously with the inoculum at a concentration of 100 kg ml-‘.
0141-0229/96/$15.00 PII SO141-0229(96)00061-O
Papers Steroid transformation 6a-Methylhydrocortisone transformation by A. glohiformis 193 cells was performed in 750-ml flasks containing 100 ml of 0.01 M Na phosphate buffer pH 7.2 on a shaker at 200 rpm and 28°C. The substrate was added to the reaction medium as a methanol solution. The final content of methanol did not exceed 5% (v/v). Preliminary results suggested that the methanol at concentrations up to 7% (v/v) does not affect the reaction kinetics. The cell concentration was 0.1-1.0 g 1-l depending on substrate concentration.
Steroid analysis The degree of 6a-methylhydrocortisone transformation into 6amethylprednisolone was determined by semiquantitative TLC on Silufol UV-254 plates or by quantitative spectrophotometry.3,’ It should be noted that a decrease in the substrate and accumulation of the product occurred stoichiometrically; the dynamics of the process was determined by product accumulation. Reaction
rate
The kinetic curves for 6a-methylhydrocortisone
transformation were obtained using weighted approximation of experimental data by cubic splines.5 The activity of cells (reaction rate) was found as the maximal first derivative along the curve. The reaction rate was expressed in units of specific activity (nmol min-’ mg-’ dry weight cells).
Oxygen consumption Oxygen consumption by cells was determined with a polarograph LP-7 (from the former Czechoslovakia) using a Clark-type platinum electrode covered with Teflon film.2 Oxygen concentration in the medium was assumed to be 250 FM. Tris phosphate buffer (50 mM) pH 7.0 was used as medium. The measurement temperature was 22-25°C. The final sample volume was 2 ml. The cell concentration in the cuvette was 1.0 g 1-l dry weight.
Results and discussion Kinetics of 6cx-methylhydrocortisone I-en-dehydrogenation Figure I shows the kinetic curves of 6a-methylhydrocortisone transformation by A. globifomzis 193 cells. As seen from the Figure, the kinetic curves of product accumulation indicate the elongation of the lag period as substrate concentration increases. Since a decrease in the substrate and accumulation of the product occurs stoichiometrically, the similar lag period is also observed on the curves of substrate consumption (not presented in the Figure). Figure 2 represents the reaction rate dependence on substrate concentration. The features of this dependence allow one to suggest the kinetics of substrate inhibition. Maximal reaction rate is observed when the introduced substrate concentration is about 1.6 mu. The sufficiently high degree of transformation (up to 99%) when substrate concentration ranged from 0.2663.192 mu and the features of kinetic curves of the reaction product accumulation allow one to exclude inhibition by the product. It should be noted that at high concentrations of 6amethylhydrocortisone exceeding the limit of solubility in water (lower than 0.266 mM>, it is difficult to determine the 502
Enzyme Microb. Technol.,
I
I
0
I
160
timeSO Figure 1 1-En-dehydrogenation of 6a-methylhydrocortisone by A. globiformiscells. Substrate concentrations were 0.266 mM (O), 0.532 mM (W), 1.064 mM (A), 1.33 mM (+ ), and 1.596 mM (v). Cell concentration was 0.5 g I-’ (dry wt). The results of spline approximation are shown by solid lines
actual concentration in transformation medium. We assume that the kinetics of substrate inhibition may be manifested more clearly with the correspondence of introduced and actual 6c+methylhydrocortisone concentrations in the transformation medium. During the study of 6c+methylhydrocortisone dehydrogenation by Arthrobacter simplex cells entrapped in karraginane geL6 the effect of substrate inhibition at substrate concentrations up to 4.522 mM was not observed. This fact does not correlate with our data at first sight. This discrepancy may be caused by different experimental conditions. In the research of Pinheiro,6 the transformation medium was supplied with menadione as an artificial electron acceptor. Menadione radically changes the kinetic mechanism of the reaction from one characteristic to the native cells having natural electron acceptors. Moreover, heated immobilized cells were used and the reaction was performed in the presence of methanol at a concentration up to 20% (v/v). Interconnection
between steroid l-en-dehydrogena-
tion and oxygen consumption In studies of hydrocortisone transformation,2.3 it was shown that microbiological dehydrogenation is accompanied by oxygen consumption and controlled by the activity of the respiratory chain. As it turns out, the transformation of the methyl derivative of hydrocortisone was also accompanied by oxygen consumption. It was interesting to investigate the interconnection between the two processes. Figure 3 presents the initial rate of oxygen consumption by bacterial cells depending on substrate concentration. The initial oxygen consumption rate grows as the 6a-methylhydrocortisone concentration increases to 1 mM. A further increase in steroid concentration results in inhibition of respiration. These data are in good agreement with the results demonstrating the change of the initial transformation rate. Notice that there is no stoichiometry between substrate conversion and oxygen consumption. This fact is probably due
1996, vol. 19, November
15
Arthrobacter V (nmole/min/mg
0
dehydrogenation
of cells, dry wt)
::::I::::I~:::I::::I:II:II/ 0 5 10
of 6 alpha-methylhydrocortisone:
25
0
20
6a-methylhydrocortisone
et al.
10
(a)
15
A. Y. Arinbasarova
25
1
2
Substrate concentration
3
4
(mM)
(mM)
l/V, min . (mg of cells, dry wt) / nmole
Figure 3 The effect of substrate concentratilon on initial rates of transformation (M) and oxygen consumption (A) by A. globiformis cells
(b)
0.25
0.20 -b 0.15 --
0.10 ;0.05 --
0
::::;::::;::::;::;, 0
1
2
3
l/mM
Figure 2 The influence of substrate concentration on reaction rate in direct (a) and double reciprocal (b) coordinates (experimental data and model solution)
to competition between 3-ketosteroid-l-endehydrogenase and endogenous substrate dehydrogenases for the respiratory chain. Figure 4 illustrates the results of the investigation of the action of Go-methylhydrocortisone on oxygen consumption by A. globiformis cells in detail. One can see that 6c+methylhydrocortisone at a concentration of 1.33 mu results in cell respiration at a twofold rate (curve 1). The subsequent addition of an oxidative phosphorylation uncoupler, carbonylcyanide-chlorophenylhydrasone (CCCP) results in a further increase in the oxygen consumption rate (curve 1); however, there is no increase in the dehydro derivative accumulation (Figure 5). Evidently, the increase in the oxygen consumption rate is due to acceleration in the oxidation of endogenous substrates. Figure 4 (curve 2) shows that 6a-methylhydrocortisone at a concentration of 2.13 mu does not stimulate respiration of cells. Moreover, it inhibits respiration caused by oxidation of endogenous substrates. It should be noted that addi-
tion of CCCP to reaction medium with substrate concentrations of 2.13 mM and higher does not result in acceleration of both oxygen consumption and steroid transformation (data not shown). Supposedly the inhibition effect of 6cr-methylhydrocortisone on the transformation process is caused by direct interaction with steroid dehydrogenase; however, this supposition does not explain the inhibition effect of steroid on the endogenous respiration of cells (Figure 4, curve 2). The absence of the CCCP accelerating effect on respiration with high steroid concentrations is also not cllear. Alternatively, the inhibition effect of steroid is due to its ability to slow down electron transfer along the respiratory chain. To verify this supposition, we also study the action of 6a-methylhydrocortisone on respiration of cells which grow without the inducer and do not have any steroid dehydrogenase activity. Data in Figure 4 show that even a low concentration of 6a-methylhydrocortisone (1.33 mu) inhibits endogenous respiration of cells (curve 4). The inhibition effect is manifested more clearly in the background of the stimulating action of CCCP (curve 5) or valinomycine as a protonophor of potassium ions (curve 6) on cell respiration. These data demonstrate directly that the inhibition effect of 6cY-methylhydrocortisone on respiration is not due to the interaction with steroid dehydrogenase but the hindering of electron transfer along the respiratory chain. It should be noted that hydrocortisone at concentrations equal to or greater than 1.33 mM reveal no inhibition effect on respiratory activity of both induced and uninduced cells (data not shown). The effect of phenazine methasulfate (PMS) and menadione on 6a-methylhydrocortisone transformation and oxygen consumption by cells grown in the presence of steroid dehydrogenase inducer was also studied. Figure 4 (curve 3) shows that while the addition of 6a-methylhydrocortisone at a concentration of 2.13 mu results in inhibition of respiration, the subsequent introduction of PMS is followed by a marked acceleration of both respiration and transformation
Enzyme Microb. Technol.,
1996, vol. 19, November
15
503
Papers Cells
63 conversion
(%)
1 42-
21-
o1
I
I
80
40 time (min)
0
Figure 5 I-En-dehydrogenation of Ga-methylhydrocortisone by A. globiformis cells in the presence of PMS ( +), menadione (A), and CCCP (W. The control is 0. The Ga-methylhydrocortsone concentration was 1.33 mM. The cell concentration was 0.7 g I--’ (dry wt); PMS and menadione were 0.1 mM; and CCCP was 5 FM.
Methylhydrocortisone, i -33 trdl
bgthylhydroccrtiscne. 1.33 mY
1 min
Figure 4 The effect of Go-methylhydrocortisone on oxygen consumption by A. globiformis cells. Concentration of cells was 1.0 g I-’ (dry wt); phenazine methasulfate (PMS) and menadione were 0.1 mM; CCCP was 5 PM; and valinomycin was 0.2 pM. Curves 1,2, and 3 correspond to the cells grown in the presence of inducer. Curves 4, 5, and 6 correspond to the cells grown without an inducer of 3-ketosteroid-I-en-dehydrogenase. Numbers on the curves indicate the respiration rate in nmoles O2 min-’ mg-’ of cells (dry wt)
of the substrate. The reaction rate increased 1.5-fold (Figure 5). Similar results were obtained with menadione. The stimulating effect of PMS and menadione on oxygen consumption and steroid transformation activity is probably caused by the fact that these artificial electron acceptors are capable of accepting the reducing equivalents from steroid dehydrogenase and transferring them directly to oxygen. In this case, the site of electron transfer inhibition by 6a-methylhydrocortisone is shunted. The above findings can also explain the lack of inhibition of 6e+methylhydrocortisone dehydrogenation by A. simplex cells in the presence of menadione.6 The absence of an acceleration in transformation after adding CCCP can be presumably considered due to a lack of control of this process by respiratory chain activity. As to the diffusion control, if the reaction rate is limited by substrate diffusion, we would observe a decrease in the lag period for product accumulation at 6c+methylhydrocortisone concentrations greater than 0.8 mu (Figure I) rather than an increase. Thus, the data show that inhibition of 6ol-methylhydrocortisone is observed on the respiratory chain probably at the level of menaquinone-cytochrome b. 504
Enzyme Microb. Technol.,
Mathematical
model
To simplify the mathematical description of the dependence of reaction rate on substrate concentration, we offer three suggestions. Firstly, the enzyme (E) can be considered a functional unit localized in a cytoplasmic membrane; it includes 3-ketosteroid-len-dehydrogenase and the respiratory chain (menaquinone and cytochromes a, b, and c). The latter regenerates an active form of the enzyme. Secondly, the above functional unit can interact with both one and several substrate molecules (S, 6a-methylhydrocortisone) forming ES and S,E complexes, respectively; the reaction rate may decrease in the latter case (multiple substrate inhibition). Lastly, the &ES complex cannot form the product P. The kinetic scheme of the reaction corresponding to the above suggestions is presented in the Figure 6. Then one can describe the reaction rate by the equation similar to Webb’s equation’:
ES
E
-
E+ P
\
//
Sn ES
\ S,E
R - + E
P + (n-1)s
Figure 6 Kinetic scheme of the reaction. E is a functional unit including 3-ketosteroid-I-en-dehydrogenase and the respiratory chain, S is the substrate (6a-methylhydrocortisone), and Pis the product (&-methylprednisolone). ES, S,E, and S,fS correspond to different types of E and S interactions
1996, vol. 19, November
15
Arthrobacter
v=v
dehydrogenation
ws+Pw, m”* 1 +
w, + w, + w,wfh
of 6 alpha-methylhydrocortisone:
(1)
where Ws = [S]/K,, W, = ([S]/yK&“, and [S] is the substrate concentration. The parameters of the model are presented in Table 1. The given values of the model parameters were determined by minimization of the function representing the sum of squares of the model solution deviations from experimental data. The simplex method and the quasi-Newton method implemented in the MINUIT system of programs’ have been used to solve the minimization problem. Figure 2 presents the model solution in direct and double inverse coordinates after data fitting. As can be seen from the Figure, the model describes the experimental data well enough, which confirms the above suppositions indirectly. Since the parameter p is not equal to zero, the reaction cannot be referred to any classical type of inhibition.7 The formation of the S,E complex could be due to binding of steroid on the cytoplasmic membrane and its modification which results in multiple substrate inhibition. Furthermore, the formation of microcrystals (or pseudocrystals) could also take place as shown for hydrocortisone earlier.9.‘o Parameter values obtained by data fitting may also explain the observed elongation of the lag period with an increase in the 6a-methylhydrocortisone concentration. Indeed, the higher 6cx-methylhydrocortisone concentration, the greater quantity of substrate molecules is bound nonspecifically with the cell membrane in a quasistationary state. The above dependence is essentially nonlinear (n % 1). As a result, the achievement of a quasistationary state characterized by the maximal rate of product formation requires a long period. It should be especially noted that the obtained values of parameters V,,,, and K, coincide with ones calculated by the linear segments of the diagram in double inverse coordinates (at a substrate concentration from 0.2661 S96 mM). It is due to the high value of parameter II and the actual lack of the inhibition effect of the substrate within stated limits.
Table 1 Parameter Vmax &I n a
Kinetic parameters value, dimension,
A. Y. Arinbasarova
et al.
The Vma,value
obtained for 6a-methylhydrocortisone (21.2 nmole min-’ mg-’ cells) is about 8 times lower than one obtained earlier for hydrocortisone (160 nmole min-’ mg-’ cells.’ This conforms with the previous supposition that the limiting stage of the process at low substrate concentrations (up to 1.6 mM) is an enzyme-substrate interaction rather than the respiratory chain activity. At the higher substrate concentrations, as mentioned above. substrate inhibition takes place at the level of the cytochrome system. As to the KM value obtained for 6ar-methylhydrocortisone, it is about 7 times higher than those obtained earlier for hydrocortisone. namely 90 yM. This fact perhaps reflects the decrease in efficiency of the ES complex formation as compared with that for hydrocortisone.
Conclusion Introduction of an additional methyl group into hydroconisone results in a change in the kinetics of microbiological dehydrogenation. The process as a whole follows substrate inhibition kinetics rather than Michaelis-Menten kinetics. The inhibition effect of 6cr-methylhydrocortisone is revealed at the level of electron transfer along the respiratory chain. The solution of the constructed model agrees with experimental data. The model parameter values after data fitting imply that multiple substrate inhibition takes place. At low substrate concentrations (up to 1.6 tnM), there is little or no inhibitory effect and the reaction follows Michaelis-Menten kinetics. The kinetic parameter values after data fitting show that it is the 6a-methylhydrocortisone interaction with 3-ketosteroid1-en-dehydrogenase that is the limiting stage for the process.
Acknowledgments The authors thank Dr. V. A. Andrjushina from the Center of Bioengineering in Russia for providing 6ol-methylhydrocortisone.
of the reaction
References
and meaning
21.2 nmole min-’ mg-’ of cells (dry wt); the maximal rate 594 PM; the dissociation constant for the ES complex 12.6; the apparent number of substrate molecules for the S,E complex formation 747; the factor characterizing the affinity change in the S,ES complex formation 0.193; the’iactor characterizing the change of rate of product formation by the S,E complex as compared to the ES complex; 4.0; the factor characterizing the change in the dissociation constant for the S,E complex as compared to the ES complex yKhrl = 2.38 mM; the dissociation constant for the .S,E complex
Spero, G. B., Thompsone, J. L., Magerlein, B. J., Hanze, A. R., Murray, H. C., Sebek, 0. K.. and Hogg. J. A. Adrenal hormones and related compounds. IV. 6-methyl steroids. .I. Am. Chem. Sot. 1956. 78, 6213-6214 Medentsev, A. G., Arinbasarova, A. Yu., Koshcheyenko, K. A., Akimenko, V. K., and Skryabin. G. K. Regulation of 3-ketosteroidI-en-dehydrogenase activity of Arthrobacter ,gfobiformis cells by a respiratory chain. J. Steroid Biochrm. 1985. 23, 365-368 Arinbasarova, A. Yu., Medentsev. A. G., Akimenko, V. K., and Koshcheyenko, K. A. Kinetic regularities of hydrocortisone dehydrogenation and oxygen uptake by free and adsorbed Arthrobacrer globiformis cells. Biochemistry (Moscow) 1984, 49, 92G927 Koshcheenko. K. A., Sukhodolskaya. G. V., Tyurin. V. S., and Skryabin, G. K. Physiological, biochemical, and morphological changes in immobilized cells during repeated periodical hydrocortisone transformations. Eur. .I. Appl. Microbial. Biotechnol. 198 I. 12. 161-169
Enzyme Microb. Technol.,
1996, vol. 19, November
15
505
Papers 5. 6.
7. 8.
506
Marchuk, G. I. Methods of Computational Muthemutics. Nauka, Moscow, 1980, 138-145 (in Russian) Pinheiro, H. M. Bioconversion of 6o-methylhydrocortisone by immobilized cells of Arthrobucter simpfe-x. MSc. thesis. Instituto Superior Tecnico, Lisboa, 1988. abstract Webb, J. L. Enzyme andMetabolic fnhibizors. Generaf Principles of Inhibition. Mir, Moscow, 1966, 70-73 (in Russian) James, F. and Roos, M. MINUIT-a system for function mini-
Enzyme Microb. Technol.,
9.
10.
1996, vol. 19, November
mization and analysis of the parameter errors and correlations. Comp. Phvs. Commun. 1975, 10,343-367 Kondo, E. and Masuo. E. Pseudo-crystallofermentation of steroid:A new process for preparing prednisolone by microorganisms. ./. Grrr. Appl. Microbial. 196 1, 7, 113-l 17 Chen, K.-C., Chang, C.-C., Chiu, C.-F., and Ling, A. C. Mathematical stimulation of pseudo-crystallofermentation of hydrocortisone by Arthrobacter simpler Biotechnol. Bioeng. 1985, 27, 253-259
15