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Substrate inhibition of rat liver and kidney arginase with fluoride
Journal of Inorganic Biochemistry 93 (2003) 243–246 www.elsevier.com / locate / jinorgbio
Substrate inhibition of rat liver and kidney arginase with fluoride Calvin D. Tormanen* Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48858, USA Received 1 August 2002; received in revised form 26 September 2002; accepted 30 September 2002
Abstract Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis–Menten kinetics. Lineweaver–Burk and Eadie–Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase. 2002 Elsevier Science Inc. All rights reserved. Keywords: Arginase; Fluoride; Substrate inhibition; Substrate activation; Rat liver and kidney
1. Introduction It has been known for many years that fluoride is an inhibitor of enzymes. A review article by Machoy [1] lists over 70 enzymes from Enzyme Commission Classes 1–4 and 6 that are inhibited by fluoride. Many of the enzymes that are inhibited by fluoride are metalloenzymes, and the fluoride ion interacts with the metal ion at the active site [2,3]. For example, fluoride inhibits the glycolytic enzyme enolase by forming a complex with the magnesium or manganese ion at the active site [4–6]. The urea cycle enzyme arginase ( L-arginine amidinohydrolase, EC 3.5.3.1) is a metalloenzyme that requires manganese ion for activity [7]. Rat liver arginase contains a binuclear Mn(II) center at the active site [8]. Inhibition of arginase by fluoride was first reported by Lenti in 1945 [9]. In 1970 Van Pilsum et al. [10] reported that rat kidney arginase was inhibited by fluoride, but the kinetics and mechanism were not studied. Recently it has been reported by Pethe et al. [11] that rat liver arginase is inhibited by fluoride by a reversible, uncompetitive mechanism. Arginase lowers the concentration of L-arginine which can reduce the amount of nitric oxide that can be produced *Tel.: 11-989-774-3252; fax: 11-989-774-3883. E-mail address: [email protected] (C.D. Tormanen).
by the enzyme nitric oxide synthase [12]. Fluoride may affect the level of nitric oxide, a potent physiological messenger, in tissues such as the penis. The purpose of this paper was to compare the inhibition of rat liver and kidney arginase by fluoride and study the substrate inhibition caused by fluoride.
2. Materials and methods
2.1. Preparation of rat liver and kidney extracts An adult male Sprague–Dawley rat was sacrificed by decapitation. The liver was removed and homogenized in 1 mM Tris buffer, pH 7.0, containing 0.154 M KCl using a Sorvall Blender. The 20% liver homogenate was centrifuged at 15 0003g for 15 min at 4 8C. The supernatant was centrifuged at 105 0003g for 60 min at 4 8C. The supernatant fraction containing cytosolic arginase was stored at 220 8C. The kidneys were removed and homogenized in 1 mM Tris buffer, pH 7.4, containing 0.25 M sucrose using a Potter–Elvehjem tissue homogenizer. The 20% kidney homogenate was centrifuged at 15 0003g for 15 min at 4 8C. The mitochondrial pellet was resuspended in 1 mM Tris buffer, pH 7.4, containing 0.25 M sucrose and 0.3% Zwittergent 3–14 surfactant at 4 8C. The surfactant-treated
0162-0134 / 02 / $ – see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00579-2
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resuspended pellet was centrifuged at 15 0003g for 15 min at 4 8C. The supernatant fraction containing the mitochondrial arginase was stored at 220 8C.
2.2. Assay of arginase activity The arginase activity was determined by measurement of L-ornithine produced as described by Tormanen [13] except that the L-arginine substrate was dissolved in 0.10 M 3-(N-morpholino)propanesulfonate (MOPS) buffer, pH 7.0. One unit of arginase activity was the formation of 1 mmol L-ornithine per hour at 37 8C. The assays were performed in duplicate.
2.3. Inhibition by fluoride Prior to assay, the liver extract was diluted from 20 to 0.03% with 0.10 M MOPS buffer, pH 7.0, and the kidney extract was diluted from 20% to 2.0% with 0.10 M MOPS buffer, pH 7.0. Potassium fluoride dihydrate was purchased from J.T. Baker (Phillipsburg, NJ). Stock solutions of potassium fluoride were made up by dilution in deionized water and stored in plastic test tubes at room temperature. For studies conducted without preincubation, 0.250 ml of substrate was added to 0.125 ml of potassium fluoride at appropriate concentration. The reaction was started by the addition of 0.125 ml of rat liver or kidney extract. The samples were incubated 10 min at 37 8C for rat liver extract and 60 min at 37 o C for rat kidney extract. The remainder of the assay was as described above. For studies conducted with preincubation, 0.125 ml of rat liver or kidney extract was added to 0.125 ml of potassium fluoride at appropriate concentrations. The samples were preincubated for 10 min at 0 8C. The reaction was started by the addition of 0.250 ml of substrate. The remainder of the assay was as described above. For kinetic studies, the concentration of L-arginine substrate was varied from 0.1 to 20 mM. Michaelis– Menten, Liverweaver–Burk, and Eadie–Hofstee plots were made from the kinetic data.
Fig. 1. Inhibition of rat liver and kidney arginase by fluoride without preincubation. 0.015% liver extract (d) and 0.5% kidney extract (s).
50% inhibition of kidney arginase occurred at about 0.4 mM KF.
3.2. Kinetics of the inhibition of rat liver arginase by fluoride As shown by the Michaelis–Menten plot in Fig. 2, fluoride caused substrate inhibition of rat liver arginase which increased as the concentration of fluoride was increased. In the absence of fluoride, liver arginase had a linear Lineweaver–Burk plot with a Km of about 2 mM L-arginine (results not shown). With L-arginine concentrations less than 4 mM, fluoride caused uncompetitive inhibition as indicated by parallel lines with Lineweaver–
3. Results
3.1. Effect of fluoride concentration on rat liver and kidney arginase activity with or without preincubation The results shown in Fig. 1 indicate that rat kidney arginase was inhibited more strongly by fluoride than liver arginase. Preincubation with fluoride did not significantly change the inhibition (results not shown). Fifty percent inhibition of liver arginase occurred at about 2 mM KF and
Fig. 2. Michaelis–Menten plot of the inhibition of rat liver arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 1 mM (s), 2 mM, (.), 4 mM (\), 8 mM (j), and 16 mM (h).
C.D. Tormanen / Journal of Inorganic Biochemistry 93 (2003) 243–246
245
Fig. 3. Michaelis–Menten plot of the inhibition of rat kidney arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 0.5 mM (s), and 1 mM (.).
Fig. 5. Eadie–Hofstee plot of the inhibition of rat kidney arginase by fluoride with preincubation. no fluoride added (d) and 1 mM potassium fluoride (s).
Burk plots (results not shown). At L-arginine concentrations above 4 mM, fluoride caused substrate inhibition of rat liver arginase (Fig. 2).
In the presence of fluoride, the Lineweaver–Burk plot for kidney arginase showed nonintersecting curved lines (Fig. 4). The inhibition appeared be of the uncompetitive type, but the substrate activation and substrate inhibition in the presence of fluoride caused nonlinearity. The substrate activation of rat kidney arginase without fluoride and the substrate inhibition with fluoride was also very apparent in the Eadie–Hofstee plot shown in Fig. 5. The steep slope with high L-arginine concentrations without fluoride indicated substrate activation. In the presence of 1 mM fluoride, the line is nearly horizontal at all concentrations of substrate (Fig. 5). Similar results were obtained with (Fig. 5) or without (results not shown) preincubation with fluoride.
3.3. Kinetics of the inhibition of rat kidney arginase by fluoride The Michaelis–Menten plot (Fig. 3) showed that fluoride also produced substrate inhibition of rat kidney arginase. In the absence of fluoride, the enzyme did not show classical Michaelis–Menten kinetics. Kidney arginase showed strong substrate activation with L-arginine concentrations above 4 mM (Fig. 3). Fluoride prevented the substrate activation of rat kidney arginase (Fig. 3).
4. Discussion
Fig. 4. Lineweaver–Burk plot of the inhibition of rat kidney arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 0.5 mM (s), 1 mM (.), 2 mM (\), and 4 mM (j).
The mechanism of inhibition of enzymes by fluoride varies depending upon the enzyme. Fluoride is a slowbinding competitive inhibitor of jack bean urease [14,15]. Fluoride is either a competitive or noncompetitive inhibitor of enolase depending upon the source and conditions [4,16–19]. Spencer and Brewer [20] have reported that fluoride is a slow binding inhibitor of yeast enolase. According to Maurer and Nowak [5] and Leboida et al. [6] fluoride replaces the hydroxide ion that is bound to the magnesium or manganese ion in the active site of yeast enolase. Fluoride is a noncompetitive inhibitor of protontranslocating ATPase of oral bacteria [21] and laccasse of Rhus [22]. Fluoride is a noncompetitive inhibitor with respect to carbon dioxide and an uncompetitive inhibitor with respect to ribulose 1,5-bisphosphate for the enzyme ribulosebisphosphate carboxylase / oxygenase [3]. Uncompetitive inhibition is rare for single substrate
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enzymes [23]. However, fluoride is an uncompetitive inhibitor of non-heme bromoperoxidase from Streptomyces aureofaciens [24], aminopeptidase from Aeromonas proteolytica [25], and Streptomyces griseus [26]. Also, the inhibition of rat liver arginase by fluoride is uncompetitive [11]. This indicates that the inhibition is due to the binding of fluoride to the arginase– L-arginine complex [11]. As with enolase [5], fluoride may be replacing the hydroxide ion that is bound to the manganese binuclear cluster [11]. The non-Michaelis–Menten kinetics found with rat kidney arginase in this report makes it difficult to determine the mechanism of inhibition by fluoride. However, when comparing small differences in substrate concentration, rat kidney arginase appears to be uncompetitively inhibited by fluoride (Fig. 4). The results shown in this paper indicated that the presence of fluoride caused substrate inhibition of both rat liver and kidney arginase (Fig. 2–5). Substrate inhibition of rat arginase has rarely been reported in the literature. Substrate inhibition of arginases from other species has been reported several times. Mora et al. [27] reported that ureotelic arginase is inhibited by substrate, but no data were provided. According to Kaysen and Strecker [28], rat liver arginase is not subject to substrate inhibition. However, they reported that rat kidney arginase is inhibited by high substrate concentrations (100–720 mM) at low pH (7.5) [27]. No substrate inhibition in the absence of fluoride was found in this study with concentrations of substrate between 0.1 and 20 mM. The results shown in this paper indicated that rat kidney arginase was subject to substrate activation by concentrations of L-arginine above 4 mM (Fig. 3–5). Kaysen and Strecker [28] first reported substrate activation of rat kidney arginase with substrate concentrations above 100 mM at pH 10. The lowest concentration of substrate used by Kaysen and Strecker was 10 mM. It is difficult to compare those results with the results that are reported in this paper because of the different substrate concentrations and pH. Neither Spector et al. [29] or Colleluori et al. [30] have reported non-Michaelis–Menten kinetics for either native or purified recombinant human kidney arginase. Therefore, the kinetics of rat kidney and human kidney arginase are different. There are no reports in the literature of substrate activation of rat liver arginase. Rat liver arginase is a trimeric protein [31]. Subunit interactions may produce multiphasic substrate binding with the liver arginase binding site dissociation constants being closer together than the kidney enzyme. Allosteric inhibition of rat liver and kidney arginase by copper and mercury ions has been reported [32]. The mutation of active site amino acid residues will be required to determine the mechanism of substrate inhibition of arginase by fluoride. The mechanism of substrate activation of rat kidney arginase can also be studied by site-directed mutagenesis. For example, site-directed mutagenesis has shown that yeast pyruvate decarboxylase has
substrate activation that requires the interaction of pyruvate with a cysteine residue located 20 angstroms from the active site thiamine pyrophosphate cofactor [33]. Rat kidney arginase may have two binding sites for substrate, a regulatory site and a catalytic site.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]
Z. Machoy, Folia Med. Cracov. 28 (1987) 61–81. T. Nowak, P.J. Maurer, Biochemistry 20 (1981) 6901–6911. T. Nilsson, R. Branden, Biochemistry 22 (1983) 1641–1645. T. Wang, A. Himoe, J. Biol. Chem. 249 (1974) 3895–3902. P.J. Maurer, T. Nowak, Biochemistry 20 (1981) 6894–6900. L. Lebioda, E. Zhang, K. Lewinski, J.M. Brewer, Proteins: Struct. Funct. Genet. 16 (1993) 219–225. C.P. Jenkinson, W.W. Grody, S.D. Cederbaum, Comp. Biochem. Physiol. 114B (1996) 107–132. R.S. Reczowski, D.E. Ash, J. Am. Chem. Soc. 114 (1992) 10992– 10994. C. Lenti, Boll. Soc. Ital. Biol. Sper. 20 (1945) 636–637. J.F. Van Pilsum, D. Taylor, B. Zakis, P. McCormick, Anal. Biochem. 35 (1970) 277–286. S. Pethe, J. Boucher, D. Mansuy, J. Inorg. Biochem. 88 (2002) 397–402. J.D. Cox, N.N. Kim, A.M. Traish, D.W. Christianson, Nat. Struct. Biol. 11 (1999) 1043–1047. C.D. Tormanen, J. Inorg. Biochem. 66 (1997) 111–118. N.E. Dixon, R.L. Blakeley, B. Zerner, Can. J. Biochem. 58 (1980) 411–488. M. Leszko, W. Zaborska, Chem. Tech. Biotechnol. 57 (1993) 113– 120. F.J. Bunick, S. Kashket, Biochemistry 21 (1982) 4285–4290. F.J. Huther, N. Psarros, H. Duschner, Infect. Immun. 58 (1990) 1043–1047. P. Bartholmes, M. Kaufmann, Caries Res. 26 (1992) 110–116. I. Kustrzeba-Wojcicka, M. Golczak, Comp. Biochem. Physiol. 126B (2000) 109–120. S.G. Spencer, J.M. Brewer, Biochem. Biophys. Res. Commun. 106 (1982) 553–558. S.V. Sutton, G.R. Bender, R.E. Marquis, Infect. Immun. 55 (1987) 2597–2603. G.B. Koudelka, M.J. Ettinger, J. Biol. Chem. 263 (1988) 3698– 3705. M. Dixon, E.C. Webb, in: Enzymes, 3rd Edition, Academic Press, New York, 1979. K. Van Pee, G. Sury, F. Lingens, Biol. Chem. Hoppe-Seyler 368 (1987) 1225–1232. G. Chen, T. Edwards, V.M. D’Souza, R.C. Holz, Biochemistry 36 (1997) 4278–4286. M.N. Harris, L. Ming, FEBS Lett. 455 (1999) 321–324. J. Mora, J. Martuscelli, J. Ortiz-Pineda, G. Soberon, Biochem. J. 96 (1965) 28–35. G.A. Kaysen, H.J. Strecker, Biochem. J. 133 (1973) 779–788. E.B. Spector, S.C.H. Rice, S. Moedjono, B. Bernard, S.D. Cederbaum, Bichem. Med. 28 (1982) 165–175. D.M. Colleluori, S.M. Morris, D.E. Ash, Arch. Biochem. Biophys. 389 (2001) 135–143. Z.F. Kanyo, L.R. Scolnick, D.E. Ash, D.W. Christianson, Nature 383 (1996) 554–557. C.D. Tormanen, J. Enzyme Inhib. 16 (2001) 443–449. J. Wang, R. Golbik, B. Seliger, M. Spinka, K. Tittmann, G. Hubner, F. Jordan, Biochemistry 40 (2001) 1755–1763.
Substrate inhibition of rat liver and kidney arginase with fluoride Calvin D. Tormanen* Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48858, USA Received 1 August 2002; received in revised form 26 September 2002; accepted 30 September 2002
Abstract Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis–Menten kinetics. Lineweaver–Burk and Eadie–Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase. 2002 Elsevier Science Inc. All rights reserved. Keywords: Arginase; Fluoride; Substrate inhibition; Substrate activation; Rat liver and kidney
1. Introduction It has been known for many years that fluoride is an inhibitor of enzymes. A review article by Machoy [1] lists over 70 enzymes from Enzyme Commission Classes 1–4 and 6 that are inhibited by fluoride. Many of the enzymes that are inhibited by fluoride are metalloenzymes, and the fluoride ion interacts with the metal ion at the active site [2,3]. For example, fluoride inhibits the glycolytic enzyme enolase by forming a complex with the magnesium or manganese ion at the active site [4–6]. The urea cycle enzyme arginase ( L-arginine amidinohydrolase, EC 3.5.3.1) is a metalloenzyme that requires manganese ion for activity [7]. Rat liver arginase contains a binuclear Mn(II) center at the active site [8]. Inhibition of arginase by fluoride was first reported by Lenti in 1945 [9]. In 1970 Van Pilsum et al. [10] reported that rat kidney arginase was inhibited by fluoride, but the kinetics and mechanism were not studied. Recently it has been reported by Pethe et al. [11] that rat liver arginase is inhibited by fluoride by a reversible, uncompetitive mechanism. Arginase lowers the concentration of L-arginine which can reduce the amount of nitric oxide that can be produced *Tel.: 11-989-774-3252; fax: 11-989-774-3883. E-mail address: [email protected] (C.D. Tormanen).
by the enzyme nitric oxide synthase [12]. Fluoride may affect the level of nitric oxide, a potent physiological messenger, in tissues such as the penis. The purpose of this paper was to compare the inhibition of rat liver and kidney arginase by fluoride and study the substrate inhibition caused by fluoride.
2. Materials and methods
2.1. Preparation of rat liver and kidney extracts An adult male Sprague–Dawley rat was sacrificed by decapitation. The liver was removed and homogenized in 1 mM Tris buffer, pH 7.0, containing 0.154 M KCl using a Sorvall Blender. The 20% liver homogenate was centrifuged at 15 0003g for 15 min at 4 8C. The supernatant was centrifuged at 105 0003g for 60 min at 4 8C. The supernatant fraction containing cytosolic arginase was stored at 220 8C. The kidneys were removed and homogenized in 1 mM Tris buffer, pH 7.4, containing 0.25 M sucrose using a Potter–Elvehjem tissue homogenizer. The 20% kidney homogenate was centrifuged at 15 0003g for 15 min at 4 8C. The mitochondrial pellet was resuspended in 1 mM Tris buffer, pH 7.4, containing 0.25 M sucrose and 0.3% Zwittergent 3–14 surfactant at 4 8C. The surfactant-treated
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resuspended pellet was centrifuged at 15 0003g for 15 min at 4 8C. The supernatant fraction containing the mitochondrial arginase was stored at 220 8C.
2.2. Assay of arginase activity The arginase activity was determined by measurement of L-ornithine produced as described by Tormanen [13] except that the L-arginine substrate was dissolved in 0.10 M 3-(N-morpholino)propanesulfonate (MOPS) buffer, pH 7.0. One unit of arginase activity was the formation of 1 mmol L-ornithine per hour at 37 8C. The assays were performed in duplicate.
2.3. Inhibition by fluoride Prior to assay, the liver extract was diluted from 20 to 0.03% with 0.10 M MOPS buffer, pH 7.0, and the kidney extract was diluted from 20% to 2.0% with 0.10 M MOPS buffer, pH 7.0. Potassium fluoride dihydrate was purchased from J.T. Baker (Phillipsburg, NJ). Stock solutions of potassium fluoride were made up by dilution in deionized water and stored in plastic test tubes at room temperature. For studies conducted without preincubation, 0.250 ml of substrate was added to 0.125 ml of potassium fluoride at appropriate concentration. The reaction was started by the addition of 0.125 ml of rat liver or kidney extract. The samples were incubated 10 min at 37 8C for rat liver extract and 60 min at 37 o C for rat kidney extract. The remainder of the assay was as described above. For studies conducted with preincubation, 0.125 ml of rat liver or kidney extract was added to 0.125 ml of potassium fluoride at appropriate concentrations. The samples were preincubated for 10 min at 0 8C. The reaction was started by the addition of 0.250 ml of substrate. The remainder of the assay was as described above. For kinetic studies, the concentration of L-arginine substrate was varied from 0.1 to 20 mM. Michaelis– Menten, Liverweaver–Burk, and Eadie–Hofstee plots were made from the kinetic data.
Fig. 1. Inhibition of rat liver and kidney arginase by fluoride without preincubation. 0.015% liver extract (d) and 0.5% kidney extract (s).
50% inhibition of kidney arginase occurred at about 0.4 mM KF.
3.2. Kinetics of the inhibition of rat liver arginase by fluoride As shown by the Michaelis–Menten plot in Fig. 2, fluoride caused substrate inhibition of rat liver arginase which increased as the concentration of fluoride was increased. In the absence of fluoride, liver arginase had a linear Lineweaver–Burk plot with a Km of about 2 mM L-arginine (results not shown). With L-arginine concentrations less than 4 mM, fluoride caused uncompetitive inhibition as indicated by parallel lines with Lineweaver–
3. Results
3.1. Effect of fluoride concentration on rat liver and kidney arginase activity with or without preincubation The results shown in Fig. 1 indicate that rat kidney arginase was inhibited more strongly by fluoride than liver arginase. Preincubation with fluoride did not significantly change the inhibition (results not shown). Fifty percent inhibition of liver arginase occurred at about 2 mM KF and
Fig. 2. Michaelis–Menten plot of the inhibition of rat liver arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 1 mM (s), 2 mM, (.), 4 mM (\), 8 mM (j), and 16 mM (h).
C.D. Tormanen / Journal of Inorganic Biochemistry 93 (2003) 243–246
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Fig. 3. Michaelis–Menten plot of the inhibition of rat kidney arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 0.5 mM (s), and 1 mM (.).
Fig. 5. Eadie–Hofstee plot of the inhibition of rat kidney arginase by fluoride with preincubation. no fluoride added (d) and 1 mM potassium fluoride (s).
Burk plots (results not shown). At L-arginine concentrations above 4 mM, fluoride caused substrate inhibition of rat liver arginase (Fig. 2).
In the presence of fluoride, the Lineweaver–Burk plot for kidney arginase showed nonintersecting curved lines (Fig. 4). The inhibition appeared be of the uncompetitive type, but the substrate activation and substrate inhibition in the presence of fluoride caused nonlinearity. The substrate activation of rat kidney arginase without fluoride and the substrate inhibition with fluoride was also very apparent in the Eadie–Hofstee plot shown in Fig. 5. The steep slope with high L-arginine concentrations without fluoride indicated substrate activation. In the presence of 1 mM fluoride, the line is nearly horizontal at all concentrations of substrate (Fig. 5). Similar results were obtained with (Fig. 5) or without (results not shown) preincubation with fluoride.
3.3. Kinetics of the inhibition of rat kidney arginase by fluoride The Michaelis–Menten plot (Fig. 3) showed that fluoride also produced substrate inhibition of rat kidney arginase. In the absence of fluoride, the enzyme did not show classical Michaelis–Menten kinetics. Kidney arginase showed strong substrate activation with L-arginine concentrations above 4 mM (Fig. 3). Fluoride prevented the substrate activation of rat kidney arginase (Fig. 3).
4. Discussion
Fig. 4. Lineweaver–Burk plot of the inhibition of rat kidney arginase by fluoride without preincubation. Various concentrations of potassium fluoride: none (d), 0.5 mM (s), 1 mM (.), 2 mM (\), and 4 mM (j).
The mechanism of inhibition of enzymes by fluoride varies depending upon the enzyme. Fluoride is a slowbinding competitive inhibitor of jack bean urease [14,15]. Fluoride is either a competitive or noncompetitive inhibitor of enolase depending upon the source and conditions [4,16–19]. Spencer and Brewer [20] have reported that fluoride is a slow binding inhibitor of yeast enolase. According to Maurer and Nowak [5] and Leboida et al. [6] fluoride replaces the hydroxide ion that is bound to the magnesium or manganese ion in the active site of yeast enolase. Fluoride is a noncompetitive inhibitor of protontranslocating ATPase of oral bacteria [21] and laccasse of Rhus [22]. Fluoride is a noncompetitive inhibitor with respect to carbon dioxide and an uncompetitive inhibitor with respect to ribulose 1,5-bisphosphate for the enzyme ribulosebisphosphate carboxylase / oxygenase [3]. Uncompetitive inhibition is rare for single substrate
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enzymes [23]. However, fluoride is an uncompetitive inhibitor of non-heme bromoperoxidase from Streptomyces aureofaciens [24], aminopeptidase from Aeromonas proteolytica [25], and Streptomyces griseus [26]. Also, the inhibition of rat liver arginase by fluoride is uncompetitive [11]. This indicates that the inhibition is due to the binding of fluoride to the arginase– L-arginine complex [11]. As with enolase [5], fluoride may be replacing the hydroxide ion that is bound to the manganese binuclear cluster [11]. The non-Michaelis–Menten kinetics found with rat kidney arginase in this report makes it difficult to determine the mechanism of inhibition by fluoride. However, when comparing small differences in substrate concentration, rat kidney arginase appears to be uncompetitively inhibited by fluoride (Fig. 4). The results shown in this paper indicated that the presence of fluoride caused substrate inhibition of both rat liver and kidney arginase (Fig. 2–5). Substrate inhibition of rat arginase has rarely been reported in the literature. Substrate inhibition of arginases from other species has been reported several times. Mora et al. [27] reported that ureotelic arginase is inhibited by substrate, but no data were provided. According to Kaysen and Strecker [28], rat liver arginase is not subject to substrate inhibition. However, they reported that rat kidney arginase is inhibited by high substrate concentrations (100–720 mM) at low pH (7.5) [27]. No substrate inhibition in the absence of fluoride was found in this study with concentrations of substrate between 0.1 and 20 mM. The results shown in this paper indicated that rat kidney arginase was subject to substrate activation by concentrations of L-arginine above 4 mM (Fig. 3–5). Kaysen and Strecker [28] first reported substrate activation of rat kidney arginase with substrate concentrations above 100 mM at pH 10. The lowest concentration of substrate used by Kaysen and Strecker was 10 mM. It is difficult to compare those results with the results that are reported in this paper because of the different substrate concentrations and pH. Neither Spector et al. [29] or Colleluori et al. [30] have reported non-Michaelis–Menten kinetics for either native or purified recombinant human kidney arginase. Therefore, the kinetics of rat kidney and human kidney arginase are different. There are no reports in the literature of substrate activation of rat liver arginase. Rat liver arginase is a trimeric protein [31]. Subunit interactions may produce multiphasic substrate binding with the liver arginase binding site dissociation constants being closer together than the kidney enzyme. Allosteric inhibition of rat liver and kidney arginase by copper and mercury ions has been reported [32]. The mutation of active site amino acid residues will be required to determine the mechanism of substrate inhibition of arginase by fluoride. The mechanism of substrate activation of rat kidney arginase can also be studied by site-directed mutagenesis. For example, site-directed mutagenesis has shown that yeast pyruvate decarboxylase has
substrate activation that requires the interaction of pyruvate with a cysteine residue located 20 angstroms from the active site thiamine pyrophosphate cofactor [33]. Rat kidney arginase may have two binding sites for substrate, a regulatory site and a catalytic site.
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