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Quantitative structure activity relationship studies on the activation of yeast AMP deaminase by polyamines
Int. J. Biochem. Vol. 19, No. 2, pp. 209-211, 1987
0020-711X/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP STUDIES ON THE ACTIVATION OF YEAST AMP DEAMINASE BY POLYAMINES MASATAKA YOSHINO1 and KEIKO MURAKAMI2 ~Department of Biochemistry, Yokohama City University School of Medicine, Yokohama 232, Japan 2Department of Laboratory Medicine, St. Marianna University School of Medicine, Kawasaki 213, Japan (Received 9 June 1986)
Abstract--l. Quantitative structure activity relationship studies on the activation of AMP deaminase by polyamines were carried out. 2. Polyamine enhanced the maximal velocity of AMP deaminase without changing the affinity for the substrate AMP. 3. Activation by polyamines of AMP deaminase can be accounted for by the simple Michaelis-Menten mechanism in the presence of ATP. 4. A close correlation between the structure and activation constants for polyamines suggests that the binding of polyamine to AMP deaminase involves primarily polar interactions.
INTRODUCTION
RESULTS AND DISCUSSION
Polyamines, which accumulate with cellular proliferation, participate in the increase in the rate of nucleic acid and protein synthesis (Abrahams and Pihl, 1981), and are responsible for the control of the activities of many enzymes (Yoshino and Murakami, 1978). Polyamines and several cations can activate A M P deaminase (EC 3.5.4.6) (Yoshino et al., 1978; Yoshino and Murakami, 1980), which acts as a control system of glycolysis (Yoshino and Murakami, 1982a,b; 1985) as well as adenylate energy charge (Chapman and Atkinson, 1973; Yoshino and Murakami, 1981), and possible role of polyamine in the stimulation of glycolysis was discussed (Yoshino and Murakami, 1982a). In order to obtain a clearer picture as to the relationship between the structure of polyamines and the action on the yeast A M P deaminase, we analyzed our results using quantitative structure activity relationships (QSAR) as developed by Hansch and co-workers (Hansch and Dunn, 1972; Hansch and Clayton, 1973). A close correlation was obtained between the degree of hydrophobicity and the activation constants of polyamines.
The effect of increasing concentrations of polyamines on the purified A M P deaminase was examined in the presence of ATP. Figure l indicated a powerful activating effect of polyamines on the enzyme; in particular, spermine showed a markedly higher affinity for the enzyme. The order of effectiveness of the polyamines as activators was spermine > spermidine > putrescine > cadaverine > 1,6-diaminohexane. We further explored the effect of polyamines on the affinity of the enzyme for the substrate A M P in the
0.10
::k >,
P,
MATERIALS AND METHODS Polyamine (rnM)
Materials. AMP and ATP were obtained from Yamasa
Co. (Tokyo, Japan). Polyamines were purchased from Sigma. All other chemicals were reagent grade. Methods. AMP deaminase was purified from the commercial baker's yeast acording to the method as described previously (Yoshino et al., 1979a). The enzyme activity was determined with the reaction mixture containing 10mM cacodylate buffer (pH 7.1), 15-25mM Na ÷, I mM ATP, various concentrations of AMP or polyamine, and the purified enzyme in a final volume of 1 or 4 ml. Ammonia liberated was determined by the method of Chaney and Marbach (1962).
Fig. 1. Effect of polyamine concentrations on the activity of AMP deaminase. The reaction mixture contained I0 mM cacodylate buffer (pH 7.1), 15mM Na ÷, 0.5mM AMP, 1 mM ATP, various concentration of polyamine, and the purified enzyme in a final volume of 1 ml. The reaction was carried out at 37°C for 5 min. Points are experimental data, and lines are theoretically drawn from equation (1) using following values of apparent K,. (O) spermine (K,, = 0.005 mM); (I-q) spermidine (K, = 0.02 mM); (A) putrescine (Ko=0.17mM); ( x ) cadaverine (Ko=0.30mM); (A) 1,6-diaminohexane (Ko = 0.65 mM).
209
MASATAKAYOSHINOand KEIKOMURAKAMI
210
E
0,10
£
steps of the substrate and polyamine, respectively. The rate constant k applies to the breakdown of E A S complex. A rate equation for the reaction scheme presented above is derived on rapid equilibrium method:
Z
[AI/Ko ~0,05
v -
VmW[S]
I +[AI/K~ K +[S]
(l)
From the assumed K, values, theoretical curves were computed and compared with the experimental ref sults. The best fit theoretical curves were obtained for > I I II 1 polyamine activation and AMP saturation relation0.5 1.0 2.0 AMP (mM) ships assuming that Ka values are 5, 20, 170, 300 Fig. 2. Effect of AMP concentration on velocity of AMP and 650#M for spermine, spermidine, putrescine, deaminase in the absence and presence of polyamine. The cadaverine and 1,6-diaminohexane, respectively in reaction mixture contained 10raM cacodylate buffer (pH the presence of 1 mM ATP (Figs 1 and 2). We now try to shed more light on the quantitative 7.1), 25mM Na ÷, 1 mM ATP, various concentration of AMP, 0.2 mM polyamine, and the enzyme in a final volume relationship between the structure and the effects of 4 ml. Points are experimental data, and lines are theor- of polyamines. We have directed our attention at a etically drawn from equation (1), using the K,~ value for parameter, the logarithm of the octanol-water parAMP of 0.28 mM. (0) no addition; (O) spermine; (Vq) tition coefficient (log P) (Hansch and Dunn, 1972; spermidine. Hansch and Clayton, 1973). The partition coefficient appears to be correlated with hydrophobic interpresence of ATP. Activation of AMP deaminase actions of ligands with the nonpolar regions of by polyamines was largely on the maximal velocity enzymes. Log P values were determined by calcuwithout alteration of S0.5and nH values (Fig. 2). These lation from the hydrophobic fragmental constants results were analyzed in terms of the following (Leo et aL, 1975). A close correlation between log P scheme, in which the saturation with AMP is hyper- and the K, values for polyamines was obtained: bolic in the presence of ATP. binding affinity of polyamines increased with decreasing hydrophobicity of these amines, suggesting that K E +S. " ES the interactions of polyamines with the enzyme involve primarily hydrophilic effects. + + A vast amount of literature has accumulated on A A the role of polyamines related to an increase in the rate of RNA synthesis as well as protein synthesis K. 11 (Abrahams and Pihl, 1981), and to the control of the activities of many enzymes (Yoshino and Murakami, K " EAS ~ EA + P EA + S . 1978). Recently, we reported that polyamines at physiological concentrations activate AMP deaminThis assumes that A is polyamine, an activator ase from rat liver (Yoshino et al., 1978) and baker's required for catalysis and that only the complex E A S yeast (Yoshino and Murakami, 1980), and AMP is active in the formation of products. The dissoci- nucleosidase from A. vinelandii (Yoshino et al., ation constants K and K, were defined for the binding 1979b). These enzymes may be important to stabilize the adenylate energy charge (Chapman and Atkinson, 1973; Schramm and Leung, 1973; Yoshino and Xt Spermlne Murakami, 1981) and be responsible for the conversion of adenine nucleotide to inosine or guanine nucleotides (Lowenstein, 1972), and AMP deaminase 2 ~ o Spermldine participates in the control of glycolytic flux (Yoshino and Murakami, 1982a). Thus, interaction of AMP deaminase with polyamines may play a principal role in the control of energy charge and of glycolysis, and 1 the synthesis of purine nucleotides during cell proliferation. A close quantitative correlation between the polyamine structure and the activation constant for each polyamine leads to the conclusion that the polar Ccldoverinee~e interactions of each polyamine with the activating Dlominohexarle site of the enzyme is essential to the conformational o I I I change of the enzyme, which causes the formation of -2 -1 0 active complex in the catalysis. The analysis of these log P Fig. 3. A correlation between the partition coefficients and interactions will serve as a guide to a more complex the activation constants for polyamines. The partition co- interaction between the enzyme and some physioet~cients were calculated from the hydrophobic fragmental logically and/or pharmacologically active agents. constants. The activation constants were taken from Fig. 1. The most fitting expression was obtained by the following Acknowledgements--This work was supported in part equation: log I/K~ = -1.403 log P -0.168 with r = 0.976. by Grants-in-Aid for Scientific Research (Nos 56570119,
Activation of AMP deaminase by polyamine 56770190, 58570150 and 61570146) from the Ministry of Education, Science and Culture of Japan. We are grateful to Professor K. Tsushima, of the Department of Biochemistry, Yokohama City University School of Medicine, for his interest and encouragement in this work.
REFERENCES
Abrahams A. K. and Pihl A. (1981) Role of polyamines in macromolecular synthesis. Trends biochem. Sci. 8, 106-107. Chaney A. L. and Marbach E. P. (1962) Modified reagents for determination of urea and ammonia. Clin. chim. Acta 8, 130-132. Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Hansch C. and Dunn W. J. (1972) Linear relationships between lipophilic character and biological activity of drugs. J. pharmac. Sci. 61, 1-19. Hansch C. and Clayton J. M. (1973) Lipophilic character and biological activity of drugs. II. The parabolic case. J. pharmac. Sci. 62, 1-21. Leo A., Jow P. Y. C., Silipo C. and Hansch C. (1975) Calculation of hydrophobic constant (log P) from 7r and f constant. J. med. Chem. 18, 865-868. Lowenstein J. M. (1972) Ammonia production in muscle and other tissues: The purine nucleotide cycle. Physiol. Rev. 52, 382-414. Schramm V. L. and Leung H. (1973) Regulation of adenosine monophosphate levels as a function of adenosine triphosphate and inorganic phosphate. A proposed metabolic role for adenosine monophosphate nucleosidase
BC
19/2--H
211
from Azotobacter vinelandii. J. biol. Chem. 248, 83138315. Yoshino M. and Murakami K. (1978) Enzymes activated by polyamines. Seikagaku 51, 1328-1334. Yoshino M. and Murakami K. (1980) Role of cations in the regulation of baker's yeast AMP deaminase. Biochim. biophys. Acta 616, 82-88. Yoshino M. and Murakami K. (1981) In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. biophys. Acta 672, 16-20. Yoshino M. and Murakami K. (1982a) AMP deaminase reaction as a control system of glycolysis in yeast. Activation of phosphofructokinase and pyruvate kinase by the AMP deaminase-ammonia system. J. biol. Chem. 257, 2822-2828. Yoshino M. and Murakami K. (1982b) AMP deaminase as a control system of glycolysis in yeast. Mechanisms of the inhibition of glycolysis by fatty acid and citrate. J. biol. Chem. 257, 10644-10649. Yoshino M. and Murakami K. (1985) AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge. J. biol. Chem. 260, 4729-4732. Yoshino M., Murakami K. and Tsushima K. (1978) The role of polyamines in the regulation of AMP deaminase isozymes. Biochim. biophys. Acta 542, 177-179. Yoshino M., Murakami K. and Tsushima K. (1979a) AMP deaminase from baker's yeast. Purification and some regulatory properties. Biochim. biophys. Acta 570, 157 166. Yoshino M., Murakami K. and Tsushima K. (1979b) Polyamines as activators of AMP nucleosidase from Azotobacter vinelandii. Experientia 35, 578-579.
0020-711X/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP STUDIES ON THE ACTIVATION OF YEAST AMP DEAMINASE BY POLYAMINES MASATAKA YOSHINO1 and KEIKO MURAKAMI2 ~Department of Biochemistry, Yokohama City University School of Medicine, Yokohama 232, Japan 2Department of Laboratory Medicine, St. Marianna University School of Medicine, Kawasaki 213, Japan (Received 9 June 1986)
Abstract--l. Quantitative structure activity relationship studies on the activation of AMP deaminase by polyamines were carried out. 2. Polyamine enhanced the maximal velocity of AMP deaminase without changing the affinity for the substrate AMP. 3. Activation by polyamines of AMP deaminase can be accounted for by the simple Michaelis-Menten mechanism in the presence of ATP. 4. A close correlation between the structure and activation constants for polyamines suggests that the binding of polyamine to AMP deaminase involves primarily polar interactions.
INTRODUCTION
RESULTS AND DISCUSSION
Polyamines, which accumulate with cellular proliferation, participate in the increase in the rate of nucleic acid and protein synthesis (Abrahams and Pihl, 1981), and are responsible for the control of the activities of many enzymes (Yoshino and Murakami, 1978). Polyamines and several cations can activate A M P deaminase (EC 3.5.4.6) (Yoshino et al., 1978; Yoshino and Murakami, 1980), which acts as a control system of glycolysis (Yoshino and Murakami, 1982a,b; 1985) as well as adenylate energy charge (Chapman and Atkinson, 1973; Yoshino and Murakami, 1981), and possible role of polyamine in the stimulation of glycolysis was discussed (Yoshino and Murakami, 1982a). In order to obtain a clearer picture as to the relationship between the structure of polyamines and the action on the yeast A M P deaminase, we analyzed our results using quantitative structure activity relationships (QSAR) as developed by Hansch and co-workers (Hansch and Dunn, 1972; Hansch and Clayton, 1973). A close correlation was obtained between the degree of hydrophobicity and the activation constants of polyamines.
The effect of increasing concentrations of polyamines on the purified A M P deaminase was examined in the presence of ATP. Figure l indicated a powerful activating effect of polyamines on the enzyme; in particular, spermine showed a markedly higher affinity for the enzyme. The order of effectiveness of the polyamines as activators was spermine > spermidine > putrescine > cadaverine > 1,6-diaminohexane. We further explored the effect of polyamines on the affinity of the enzyme for the substrate A M P in the
0.10
::k >,
P,
MATERIALS AND METHODS Polyamine (rnM)
Materials. AMP and ATP were obtained from Yamasa
Co. (Tokyo, Japan). Polyamines were purchased from Sigma. All other chemicals were reagent grade. Methods. AMP deaminase was purified from the commercial baker's yeast acording to the method as described previously (Yoshino et al., 1979a). The enzyme activity was determined with the reaction mixture containing 10mM cacodylate buffer (pH 7.1), 15-25mM Na ÷, I mM ATP, various concentrations of AMP or polyamine, and the purified enzyme in a final volume of 1 or 4 ml. Ammonia liberated was determined by the method of Chaney and Marbach (1962).
Fig. 1. Effect of polyamine concentrations on the activity of AMP deaminase. The reaction mixture contained I0 mM cacodylate buffer (pH 7.1), 15mM Na ÷, 0.5mM AMP, 1 mM ATP, various concentration of polyamine, and the purified enzyme in a final volume of 1 ml. The reaction was carried out at 37°C for 5 min. Points are experimental data, and lines are theoretically drawn from equation (1) using following values of apparent K,. (O) spermine (K,, = 0.005 mM); (I-q) spermidine (K, = 0.02 mM); (A) putrescine (Ko=0.17mM); ( x ) cadaverine (Ko=0.30mM); (A) 1,6-diaminohexane (Ko = 0.65 mM).
209
MASATAKAYOSHINOand KEIKOMURAKAMI
210
E
0,10
£
steps of the substrate and polyamine, respectively. The rate constant k applies to the breakdown of E A S complex. A rate equation for the reaction scheme presented above is derived on rapid equilibrium method:
Z
[AI/Ko ~0,05
v -
VmW[S]
I +[AI/K~ K +[S]
(l)
From the assumed K, values, theoretical curves were computed and compared with the experimental ref sults. The best fit theoretical curves were obtained for > I I II 1 polyamine activation and AMP saturation relation0.5 1.0 2.0 AMP (mM) ships assuming that Ka values are 5, 20, 170, 300 Fig. 2. Effect of AMP concentration on velocity of AMP and 650#M for spermine, spermidine, putrescine, deaminase in the absence and presence of polyamine. The cadaverine and 1,6-diaminohexane, respectively in reaction mixture contained 10raM cacodylate buffer (pH the presence of 1 mM ATP (Figs 1 and 2). We now try to shed more light on the quantitative 7.1), 25mM Na ÷, 1 mM ATP, various concentration of AMP, 0.2 mM polyamine, and the enzyme in a final volume relationship between the structure and the effects of 4 ml. Points are experimental data, and lines are theor- of polyamines. We have directed our attention at a etically drawn from equation (1), using the K,~ value for parameter, the logarithm of the octanol-water parAMP of 0.28 mM. (0) no addition; (O) spermine; (Vq) tition coefficient (log P) (Hansch and Dunn, 1972; spermidine. Hansch and Clayton, 1973). The partition coefficient appears to be correlated with hydrophobic interpresence of ATP. Activation of AMP deaminase actions of ligands with the nonpolar regions of by polyamines was largely on the maximal velocity enzymes. Log P values were determined by calcuwithout alteration of S0.5and nH values (Fig. 2). These lation from the hydrophobic fragmental constants results were analyzed in terms of the following (Leo et aL, 1975). A close correlation between log P scheme, in which the saturation with AMP is hyper- and the K, values for polyamines was obtained: bolic in the presence of ATP. binding affinity of polyamines increased with decreasing hydrophobicity of these amines, suggesting that K E +S. " ES the interactions of polyamines with the enzyme involve primarily hydrophilic effects. + + A vast amount of literature has accumulated on A A the role of polyamines related to an increase in the rate of RNA synthesis as well as protein synthesis K. 11 (Abrahams and Pihl, 1981), and to the control of the activities of many enzymes (Yoshino and Murakami, K " EAS ~ EA + P EA + S . 1978). Recently, we reported that polyamines at physiological concentrations activate AMP deaminThis assumes that A is polyamine, an activator ase from rat liver (Yoshino et al., 1978) and baker's required for catalysis and that only the complex E A S yeast (Yoshino and Murakami, 1980), and AMP is active in the formation of products. The dissoci- nucleosidase from A. vinelandii (Yoshino et al., ation constants K and K, were defined for the binding 1979b). These enzymes may be important to stabilize the adenylate energy charge (Chapman and Atkinson, 1973; Schramm and Leung, 1973; Yoshino and Xt Spermlne Murakami, 1981) and be responsible for the conversion of adenine nucleotide to inosine or guanine nucleotides (Lowenstein, 1972), and AMP deaminase 2 ~ o Spermldine participates in the control of glycolytic flux (Yoshino and Murakami, 1982a). Thus, interaction of AMP deaminase with polyamines may play a principal role in the control of energy charge and of glycolysis, and 1 the synthesis of purine nucleotides during cell proliferation. A close quantitative correlation between the polyamine structure and the activation constant for each polyamine leads to the conclusion that the polar Ccldoverinee~e interactions of each polyamine with the activating Dlominohexarle site of the enzyme is essential to the conformational o I I I change of the enzyme, which causes the formation of -2 -1 0 active complex in the catalysis. The analysis of these log P Fig. 3. A correlation between the partition coefficients and interactions will serve as a guide to a more complex the activation constants for polyamines. The partition co- interaction between the enzyme and some physioet~cients were calculated from the hydrophobic fragmental logically and/or pharmacologically active agents. constants. The activation constants were taken from Fig. 1. The most fitting expression was obtained by the following Acknowledgements--This work was supported in part equation: log I/K~ = -1.403 log P -0.168 with r = 0.976. by Grants-in-Aid for Scientific Research (Nos 56570119,
Activation of AMP deaminase by polyamine 56770190, 58570150 and 61570146) from the Ministry of Education, Science and Culture of Japan. We are grateful to Professor K. Tsushima, of the Department of Biochemistry, Yokohama City University School of Medicine, for his interest and encouragement in this work.
REFERENCES
Abrahams A. K. and Pihl A. (1981) Role of polyamines in macromolecular synthesis. Trends biochem. Sci. 8, 106-107. Chaney A. L. and Marbach E. P. (1962) Modified reagents for determination of urea and ammonia. Clin. chim. Acta 8, 130-132. Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate energy charge by the adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Hansch C. and Dunn W. J. (1972) Linear relationships between lipophilic character and biological activity of drugs. J. pharmac. Sci. 61, 1-19. Hansch C. and Clayton J. M. (1973) Lipophilic character and biological activity of drugs. II. The parabolic case. J. pharmac. Sci. 62, 1-21. Leo A., Jow P. Y. C., Silipo C. and Hansch C. (1975) Calculation of hydrophobic constant (log P) from 7r and f constant. J. med. Chem. 18, 865-868. Lowenstein J. M. (1972) Ammonia production in muscle and other tissues: The purine nucleotide cycle. Physiol. Rev. 52, 382-414. Schramm V. L. and Leung H. (1973) Regulation of adenosine monophosphate levels as a function of adenosine triphosphate and inorganic phosphate. A proposed metabolic role for adenosine monophosphate nucleosidase
BC
19/2--H
211
from Azotobacter vinelandii. J. biol. Chem. 248, 83138315. Yoshino M. and Murakami K. (1978) Enzymes activated by polyamines. Seikagaku 51, 1328-1334. Yoshino M. and Murakami K. (1980) Role of cations in the regulation of baker's yeast AMP deaminase. Biochim. biophys. Acta 616, 82-88. Yoshino M. and Murakami K. (1981) In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts. Biochim. biophys. Acta 672, 16-20. Yoshino M. and Murakami K. (1982a) AMP deaminase reaction as a control system of glycolysis in yeast. Activation of phosphofructokinase and pyruvate kinase by the AMP deaminase-ammonia system. J. biol. Chem. 257, 2822-2828. Yoshino M. and Murakami K. (1982b) AMP deaminase as a control system of glycolysis in yeast. Mechanisms of the inhibition of glycolysis by fatty acid and citrate. J. biol. Chem. 257, 10644-10649. Yoshino M. and Murakami K. (1985) AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge. J. biol. Chem. 260, 4729-4732. Yoshino M., Murakami K. and Tsushima K. (1978) The role of polyamines in the regulation of AMP deaminase isozymes. Biochim. biophys. Acta 542, 177-179. Yoshino M., Murakami K. and Tsushima K. (1979a) AMP deaminase from baker's yeast. Purification and some regulatory properties. Biochim. biophys. Acta 570, 157 166. Yoshino M., Murakami K. and Tsushima K. (1979b) Polyamines as activators of AMP nucleosidase from Azotobacter vinelandii. Experientia 35, 578-579.