Eur. J. Biochem. 65, 271 -274 (1976)
Glutamine Transaminase from Brain Tissue Further Studies on Kinetic Properties and Specificity of the Enzyme Freddy VAN LEUVEN Laboratory of Normal and Pathological Physiology, University of Ghent (Received December 9, 19?5/March 4, 1976)
Glutamine transaminase, highly purified from rat brain, was studied. In the first series of experiments, the kinetics of the transamination reaction between 2-oxoglutaramate and phenylalanine were examined in order to determine the type of reaction mechanism. This proved to be of the pingpong type, as can be expected for a transamination. The specificity of the enzyme for various amino acids and 2-0x0 acids was then studied in detail. The most active substrates were glutamine, methionine and ethionine as amino-group donors, and phenylpyruvate, glyoxalate and 2-oxo-4-methiolbutyrate as amino-group acceptors. For these and several other substrates, the kinetic constants, V and K,, were determined.
In a previous communication we described the purification of glutamine : 2-oxo-acid transaminase from rat brain tissue and some physical and catalytic properties of this highly-purified enzyme [l]. In view of the importance which this enzyme might have in the metabolism and synthesis of amino acids in the brain, further experiments on the kinetics and specificity of glutamine transaminase of brain tissue were done. A kinetic analysis of the transamination reaction was made, and 30 amino acids and 17 0x0 acids were examined for their ability to participate in the transamination reaction. The effect of substrate concentration on the initial reaction rate was studied for the more important of the substrates, and the kinetic constants, K , and V , were calculated in order to get a better understanding of the affinity of the enzyme for various substrates.
MATERIALS AND METHODS Glutamine transaminase from rat brain was purified according to a previously described procedure [I]. The enzyme activity and initial reaction rates were determined with previously described techniques [1,2]. Only linear parts of the reaction progress curves were taken to determine initial reaction rates. The substrates were commercial products of the highest purity available and were purchased from Sigma Chemical Co. and Calbiochem, with the exEnzyme. Glutamine transaminase or L-glutamine: 2-oxoacid aininotransferase (EC 2.6.1.1 5).
ception of 2-oxoglutaramate (synthesized according to Meister [3]), 3-mercaptopyruvate (prepared according to Parrod [4]) and 5-N-methylglutamine (prepared according to Lichtenstein [5]). RESULTS
Kinetics of' the Reaction In order to analyse the type of reaction mechanism, the reaction of 2-oxoglutaramate with phenylalanine was studied in detail. According to the procedure of Velick and Vavra [6], the initial velocity of the reaction was measured as a function of the concentration of phenylalanine at four fixed concentrations of the second substrate, 2-oxoglutaramate. The primary plot (Fig.1) of these reactions showed a set of parallel straight lines. When 2-oxoglutaramate was the variable substrate at fixed phenylalanine concentrations, a comparable set of parallel lines was obtained. Fig.2 represents the points of intersection of these lines with the ordinate, plotted against the reciprocals of the concentration of the second substrate. Both possible combinations are shown. As can be seen, a linear relationship was obtained and both lines have a common intersection point on the ordinate. This represents the reciprocal of the maximal velocity of the reaction. The value calculated from these data was 96.5 k 3.6 pmol h-' mg protein-'. The Michaelis constants calculated were 21.0 f 0.8 mM for 2-oxoglutaramate, and 0.092 f 0.007 mM for phenylalanine. The graphic plot of the results indicates that the reaction proceeds according to a reaction mechanism
272
Brain Glutamine Transaminase [2-0xoglutararnate] (rnM)
,
0.03
I , , , , , 2 4 6 8 1 0
'0
[Phenylalaninel-' (rnM-')
"0
2
4 6 [Phenylalan I ne].'
8 (rnM-')
10
Fig. 1. Primary plot oJ the reaction of2-o.woglutarumate with phenyluluntne. The initial reaction rate ( u ) is expressed in vmol enolborate complex of phenylpyruvate formed per h per mg protein
0.03
1
[Phenylalaninel-'
'0
(rnM-')
0.02 0.04 0.06 0.08 0.10 [2-0xoglutararnate]-' (rnM-')
Fig. 2. Secondary plot of'the data of'Fig. I . The intercepts of the straight lines with the ordinate are replotted against the reciprocal of the concentration of 2-oxoglutaramate (a).The line corresponding to the open symbols (0)was obtained by the procedure described in the text. V represents the values of the intercepts in the primary plot
of the ping-pong type [7]. This was confirmed by another experiment in which the concentrations of the two substrates were varied in a constant ratio to each other. The double-reciprocal plot of these results is linear (Fig. 3), and this line intersects with the ordinate at the point given by the reciprocal value of the maximal velocity. The value calculated from these data was 94.5 3.2 pmol h-' mg protein-' which is in excellent agreement with the value obtained in the experiments described above.
Fig. 3. Ejjbct on initial velocity of' the reuction when the concentrations of phenylalanine and 2-oxoglutaramate are varied in a consiant ratio to each other. The concentration of 2-oxoglutaramate was 100-fold higher than that of phenylalanine
Specificity of the Enzyme for Amino Acids The amino acids were tested with phenylpyruvate or glyoxalate as the amino acceptor. The results are presented in Table 1, where the rate of transamination is shown relative to the rate of the ghtdmine-phenylpyruvate or glutamine-glyoxalate reactions. As can be seen from Table 1, the natural amino acid methionine is very effective as an amino donor in the transamination reaction, with a rate that exceeds that of glutamine by about 30 %. Phenylalanine is another natural amino acid with properties of a substrate for the enzyme, although it reacts at a much slower rate. Analogues of glutamine and methionine showed similar activities of variable degree. The effect of the amino acid concentration on the initial reaction rate was studied for the most active substrates', with phenylpyruvate as the second substrate at constant concentration (0.36 mM). The kinetic constants calculated from these date are summarized in Table 2.
Specificity ofthe Enzyme f o r 0x0 Acids The initial reaction rate of a number of 0x0 acids with glutamine was determined. The results of these experiments are shown in Table 3. The initial reaction rate is expressed relative to the rate of the phenylpyruvate-glutamine reaction. With the exception of 3-bromopyruvate, all of the 0x0 acids are more or less active as an amino group acceptor in the transamination reaction with glutamine and are thus substrates for brain glutamine transaminase. For the most active 0x0 acids1, the influence of substrate concentration on the initial reaction rate It was technically impossible to get accurate results with the less active substrates.
F. Van Leuven
213
Table 1. Relative activiiy of glutumine transamit~aseagainst various amino wid.< The initial reaction rates are expressed relative to the rate for the glutamine-phenylpyr~~vate and the glutamine-glyoxalate reactions. The initial velocities were determined by measuring the disappearance of the enol-borate complex of phenylpyruvate and by using [U-14C]glyoxalate. Other amino acids tested at 10 m M but found not to be active were L-glutamate, L-aspartate, 1.-cystein, L-glycine, L-alanine, L-serine, L-proline, L-hydroxyproline, L-valine, L-leucine, L-isoleucine, L-threonine, L-lysine, and 4-aminobutyrate Amino acid
Concentration
L-Glutamine L-Methionine 1.-Ethionine L-S-Methylcysteine L-Methionine ( S R ) sulfoxide Ill-Methionine ( S R ) sulfoximine L-5-N-Hydroxyglutaminc L-5-N-Meth ylglutatnine ~-Glutamate-5-methylester ~-Glutamate-5-ethylester L-Cysteine L-Aspardgine L-Phenylalanine L-Tyrosine L- H istidine L-Tryptophan
Relative rate with phenyl- glyoxalate Dvruvate
mM
7i
10 5 5 10 10 20 20 20 40 40 10 10 2 2 2 2
(100) 127 112
(100) 131 108
98 46 34 97 29 31 14 4 2
-
34 -
Table 3. Relative uctivity of gfuramine transuminusc. againAt vurious 2-o.ro nc,ids The initial reaction rates are expressed relative to the rate for glutamine-phenylpyruvate reaction. [U-14C]Glutamine was used to determine the initial reaction rates 0 x 0 acid
Phenylpyruvate Glyoxalate 2-Oxo-4-methiohtyrate p-Hydroxyphenylpyruvate 3-Mercaptopyruvate Pyruvate 2-Oxobutyrate 2-Oxovalerate 2-0x0-isovalerate 2-0x0-3-methyl valerate 2-0x0-4-methyl valerate 3-Hydroxypyruvate 3-Bromopyruvate 3-Indolepyruvate Oxaloacetate 2-Oxoglutarate
~
-
14 11 9 1 2
~
KM
V
mM
pmol h-' mg protein-'
0.63 i- 0.01 1.96 0.10 2.53 & 0.14 2.85 k 0.13 16.5 k 1.2 96.0 k 10 6.91 k 0.37 2.15 f 0.06 0.092 i 0.007
+
* **
DISCUSSION The kinetic analysis of the reaction 2-Oxoglutaramate + phenylalanine Glutamine phenylpyruvate
+
5 1 10 20 20 20 20 20 20 20 20 1 10 10
Table 4. Kinetic parametersf o r the t~un.~amination oj'Z-o.~oat.id.7 [U-'4C]Glutamine (10 mM) was used as the amino group donor except for 2-oxoglutaramate, where phenylalanine (1 mM) was used. Values standard deviation are shown
Glyoxalate 2-0x0-4-methiolbutyrate 3-Mercaptopyruvate Pyruvate 2-Oxobutyrate 2-Oxovalerate 2-Oxoglutaramate P henylpyruvate
K,
V
mM
pmol h - ' mgprotein
1.46 k 0.06 0.25 & 0.01 0.61 k 0.41 13.5 & 0.8 9.19 k 0.48 0.52 6.76 20.5 0.9 0.023 k 0.001
300 k 456 k 239 84+ 108 98k 87+ I76 &
+ +
* *
'
8 15 14 4 5 7 3 7
+
165 3 217 & 11 12 220 167 & 7 64 4 7 63 63 & 3 86 k 2 97 i 4
was studied, and the kinetic parameters calculated from these date are presented in Table 4.
+
100 146 235 89 71 28 37 42 12 8 32 33 0 18 18 7
-
Table 2. Kinetic parameters .for the trunsamination of' umino acids The amino acceptor was phenylpyruvate (0.36 mM) except for L-phenylalanine, where 2-oxoglutaramate (100 mM) was used. With each value the standard deviation is shown. The disappearance or the formation of the end-borate complex of phenylpyruvate was measured
r-Glutamine L-Methionine L-Ethionine 1.-S-Methylcysteine ~-Glutamate-5-methylester ~-Glutamate-5-ethylester ~.-5-N-MethyIglutamine L-Methionine (SR)sulfoxide L-Phenylalanine
mM 1 10
-
0 x 0 acid
Amino acid
Concentration Relative rate with glutamine
is consistent with a ping-pong reaction mechanism, as can be expected for a transamination reaction. The kinetic parameters, K , and V , were calculated according the equation 1 1 v v a h with K, and Kh representing the Michaelis constants of both substrates, and a and b their concentration. When these concentrations are kept at a constant ratio (e.g. h = 100 a),the equation becomes:
The plot (Fig. 3) is in accordance with this equation, as is demonstrated by the excellent agreement of the
F. Van Leuven : Brain Glutamine Transaminase
274
values for the maximal reaction rate obtained in this and the first experiment. The Michaelis constant for 2-oxoglutaramate is rather high, but it should be remembered that this 0x0 acid undergoes spontaneous cyclization. The concentration of the open form, which is probably the reactive molecule in the transamination reaction, amounts to only 0.3'j/, of the total concentration in solution (at neutral pH and 30 "C) [8]. Assuming the same value under the conditions of our experiments, a value of 0.06 mM for the K, of 2-oxoglutaramate is obtained. Although this value may only be an approximation, it probably better reflects the real value for the Michaelis constant of this 0x0 acid. This reasoning holds, provided the interconversion of the cyclic and open chain isomers of 2-0x0glutaramate is not rate-limiting relative to the transamination reaction, which is the case at the high pH in our experiments [8]. The specificity of glutamine transaminase was determined with 30 amino acids. Of the natural amino acids, methionine proved to be a better substrate than glutamine itself. Phenylalanine and tyrosine were also active in the transamination with glyoxalate, as were cysteine and histidine, but at a much slower rate. The glutamine analogues, 5-N-methylglutamine and 5-N-hydroxyglutamine, and the 5-methyl and 5-ethyl esters of glutamic acid, are also substrates for the purified enzyme. The analogues of methionine are even better substrates. That methionine is probably transaminated by the same enzyme as glutamine is indicated by the fact that the maximal velocity of the reaction of glutamine with phenylpyruvate is not changed by the addition of methionine (Fig. 4). The kinetic constants, determined for the most active amino acids, show that the affinity of the enzyme for glutamine is greater than for methionine (Table 2). Considering the fact that the concentration of glutamine in the brain of the rat is much higher than that of methionine, we may speculate that in vivo glutamine is the normal substrate of the enzyme. Of the 0x0 acids that were studied, all showed some activity with the enzyme. The most important were phenylpyruvate, glyoxalate and the 0x0 analogues of tyrosine, methionhe, cysteine, and glutamine. These data indicate that some 0x0 acids are substrates for glutamine transaminase, while the corresponding amino acids are not (at least in our experimental conditions). This means that the enzyme is more specific for the amino acids than for the 0x0 acid substrate. which was also observed for the
0' 0
I
1
I
I
I
2 3 4 [Glutarnine]-' (rnM-')
I
5
Fig.4. Influence of the concrwtrarion of glutaminr on the initiul reaction rule with phenylpyruvate and the effict of methionine. The initial rate was determined with glutamine alone ( 0 ) and with The concentration glutamine plus methionine (1.25 mM) added (0). of phenylpyruvate was measured to determine initial reaction rates
enzyme obtained from sources other than brain [9, lo]. This phenomenon can not be readily explained. When the K,,, values of glutamine, phenylalanine and methionine are compared with those for the corresponding 0x0 analogues, one observes that the enzyme has a greater affinity for the 0x0 acids than for the amino acids. This can be important in that the 0x0 acids will more readily be bound by the enzyme. Although it is an intriguing observation, it is clear that the less stringent specificity of the enzyme for the 0x0 acid substrate is important in vivo. Many 0x0 acids can be aminated by this reaction, with the formation of the corresponding amino acids. Moreover, the excess of ammonia toxic for the brain and stored in glutamine is used in the synthesis of several important compounds.
REFERENCES 1. Van Leuven, F. (1975) Eur. J . Biochem. 58, 153-158. 2. Van Leuven, F. (1974) Arch. Int. Physiol. Biochem. 82, 219283. 3. Meister, A. (1953) .J. Bid. Chcm. 200, 571 -589. 4. Parrod, J . (1942) C. R. Hc>hd.SPances Acad. Sci. (Puri,s) 251. 146- 148. 64, 1021 - 1022. 5. Lichtenstein, N. (1942) J . Am. C'hem. SOC. 6 . Velick, S. F. & Vavra. J. (1962) J . B i d . Chem. 237,2109- 2122. I. Cleland, W . W. (1963) Biochim. Bioph.vs. Acta, 67, 104- 137. 8. Hersh, L. B. (1971) Biochemistry, 10, 2884-2891. 9. Cooper, A. J. L. & Meister, A. (1972) Biochemistry, 11, 661 671. 10. Cooper, A. J. L. & Meister, A. (1974) J . Bid. Chem. 240, 2554- 2561.
F. Van Leuven, Laboratoriuin voor Normale en Pathologische Fysiologie, Fakulteit voor Geneeskunde, Rijksuniversiteit te Gent, De Pintelaan 135, 8-9000 Gent, Belgium
~
Glutamine Transaminase from Brain Tissue Further Studies on Kinetic Properties and Specificity of the Enzyme Freddy VAN LEUVEN Laboratory of Normal and Pathological Physiology, University of Ghent (Received December 9, 19?5/March 4, 1976)
Glutamine transaminase, highly purified from rat brain, was studied. In the first series of experiments, the kinetics of the transamination reaction between 2-oxoglutaramate and phenylalanine were examined in order to determine the type of reaction mechanism. This proved to be of the pingpong type, as can be expected for a transamination. The specificity of the enzyme for various amino acids and 2-0x0 acids was then studied in detail. The most active substrates were glutamine, methionine and ethionine as amino-group donors, and phenylpyruvate, glyoxalate and 2-oxo-4-methiolbutyrate as amino-group acceptors. For these and several other substrates, the kinetic constants, V and K,, were determined.
In a previous communication we described the purification of glutamine : 2-oxo-acid transaminase from rat brain tissue and some physical and catalytic properties of this highly-purified enzyme [l]. In view of the importance which this enzyme might have in the metabolism and synthesis of amino acids in the brain, further experiments on the kinetics and specificity of glutamine transaminase of brain tissue were done. A kinetic analysis of the transamination reaction was made, and 30 amino acids and 17 0x0 acids were examined for their ability to participate in the transamination reaction. The effect of substrate concentration on the initial reaction rate was studied for the more important of the substrates, and the kinetic constants, K , and V , were calculated in order to get a better understanding of the affinity of the enzyme for various substrates.
MATERIALS AND METHODS Glutamine transaminase from rat brain was purified according to a previously described procedure [I]. The enzyme activity and initial reaction rates were determined with previously described techniques [1,2]. Only linear parts of the reaction progress curves were taken to determine initial reaction rates. The substrates were commercial products of the highest purity available and were purchased from Sigma Chemical Co. and Calbiochem, with the exEnzyme. Glutamine transaminase or L-glutamine: 2-oxoacid aininotransferase (EC 2.6.1.1 5).
ception of 2-oxoglutaramate (synthesized according to Meister [3]), 3-mercaptopyruvate (prepared according to Parrod [4]) and 5-N-methylglutamine (prepared according to Lichtenstein [5]). RESULTS
Kinetics of' the Reaction In order to analyse the type of reaction mechanism, the reaction of 2-oxoglutaramate with phenylalanine was studied in detail. According to the procedure of Velick and Vavra [6], the initial velocity of the reaction was measured as a function of the concentration of phenylalanine at four fixed concentrations of the second substrate, 2-oxoglutaramate. The primary plot (Fig.1) of these reactions showed a set of parallel straight lines. When 2-oxoglutaramate was the variable substrate at fixed phenylalanine concentrations, a comparable set of parallel lines was obtained. Fig.2 represents the points of intersection of these lines with the ordinate, plotted against the reciprocals of the concentration of the second substrate. Both possible combinations are shown. As can be seen, a linear relationship was obtained and both lines have a common intersection point on the ordinate. This represents the reciprocal of the maximal velocity of the reaction. The value calculated from these data was 96.5 k 3.6 pmol h-' mg protein-'. The Michaelis constants calculated were 21.0 f 0.8 mM for 2-oxoglutaramate, and 0.092 f 0.007 mM for phenylalanine. The graphic plot of the results indicates that the reaction proceeds according to a reaction mechanism
272
Brain Glutamine Transaminase [2-0xoglutararnate] (rnM)
,
0.03
I , , , , , 2 4 6 8 1 0
'0
[Phenylalaninel-' (rnM-')
"0
2
4 6 [Phenylalan I ne].'
8 (rnM-')
10
Fig. 1. Primary plot oJ the reaction of2-o.woglutarumate with phenyluluntne. The initial reaction rate ( u ) is expressed in vmol enolborate complex of phenylpyruvate formed per h per mg protein
0.03
1
[Phenylalaninel-'
'0
(rnM-')
0.02 0.04 0.06 0.08 0.10 [2-0xoglutararnate]-' (rnM-')
Fig. 2. Secondary plot of'the data of'Fig. I . The intercepts of the straight lines with the ordinate are replotted against the reciprocal of the concentration of 2-oxoglutaramate (a).The line corresponding to the open symbols (0)was obtained by the procedure described in the text. V represents the values of the intercepts in the primary plot
of the ping-pong type [7]. This was confirmed by another experiment in which the concentrations of the two substrates were varied in a constant ratio to each other. The double-reciprocal plot of these results is linear (Fig. 3), and this line intersects with the ordinate at the point given by the reciprocal value of the maximal velocity. The value calculated from these data was 94.5 3.2 pmol h-' mg protein-' which is in excellent agreement with the value obtained in the experiments described above.
Fig. 3. Ejjbct on initial velocity of' the reuction when the concentrations of phenylalanine and 2-oxoglutaramate are varied in a consiant ratio to each other. The concentration of 2-oxoglutaramate was 100-fold higher than that of phenylalanine
Specificity of the Enzyme for Amino Acids The amino acids were tested with phenylpyruvate or glyoxalate as the amino acceptor. The results are presented in Table 1, where the rate of transamination is shown relative to the rate of the ghtdmine-phenylpyruvate or glutamine-glyoxalate reactions. As can be seen from Table 1, the natural amino acid methionine is very effective as an amino donor in the transamination reaction, with a rate that exceeds that of glutamine by about 30 %. Phenylalanine is another natural amino acid with properties of a substrate for the enzyme, although it reacts at a much slower rate. Analogues of glutamine and methionine showed similar activities of variable degree. The effect of the amino acid concentration on the initial reaction rate was studied for the most active substrates', with phenylpyruvate as the second substrate at constant concentration (0.36 mM). The kinetic constants calculated from these date are summarized in Table 2.
Specificity ofthe Enzyme f o r 0x0 Acids The initial reaction rate of a number of 0x0 acids with glutamine was determined. The results of these experiments are shown in Table 3. The initial reaction rate is expressed relative to the rate of the phenylpyruvate-glutamine reaction. With the exception of 3-bromopyruvate, all of the 0x0 acids are more or less active as an amino group acceptor in the transamination reaction with glutamine and are thus substrates for brain glutamine transaminase. For the most active 0x0 acids1, the influence of substrate concentration on the initial reaction rate It was technically impossible to get accurate results with the less active substrates.
F. Van Leuven
213
Table 1. Relative activiiy of glutumine transamit~aseagainst various amino wid.< The initial reaction rates are expressed relative to the rate for the glutamine-phenylpyr~~vate and the glutamine-glyoxalate reactions. The initial velocities were determined by measuring the disappearance of the enol-borate complex of phenylpyruvate and by using [U-14C]glyoxalate. Other amino acids tested at 10 m M but found not to be active were L-glutamate, L-aspartate, 1.-cystein, L-glycine, L-alanine, L-serine, L-proline, L-hydroxyproline, L-valine, L-leucine, L-isoleucine, L-threonine, L-lysine, and 4-aminobutyrate Amino acid
Concentration
L-Glutamine L-Methionine 1.-Ethionine L-S-Methylcysteine L-Methionine ( S R ) sulfoxide Ill-Methionine ( S R ) sulfoximine L-5-N-Hydroxyglutaminc L-5-N-Meth ylglutatnine ~-Glutamate-5-methylester ~-Glutamate-5-ethylester L-Cysteine L-Aspardgine L-Phenylalanine L-Tyrosine L- H istidine L-Tryptophan
Relative rate with phenyl- glyoxalate Dvruvate
mM
7i
10 5 5 10 10 20 20 20 40 40 10 10 2 2 2 2
(100) 127 112
(100) 131 108
98 46 34 97 29 31 14 4 2
-
34 -
Table 3. Relative uctivity of gfuramine transuminusc. againAt vurious 2-o.ro nc,ids The initial reaction rates are expressed relative to the rate for glutamine-phenylpyruvate reaction. [U-14C]Glutamine was used to determine the initial reaction rates 0 x 0 acid
Phenylpyruvate Glyoxalate 2-Oxo-4-methiohtyrate p-Hydroxyphenylpyruvate 3-Mercaptopyruvate Pyruvate 2-Oxobutyrate 2-Oxovalerate 2-0x0-isovalerate 2-0x0-3-methyl valerate 2-0x0-4-methyl valerate 3-Hydroxypyruvate 3-Bromopyruvate 3-Indolepyruvate Oxaloacetate 2-Oxoglutarate
~
-
14 11 9 1 2
~
KM
V
mM
pmol h-' mg protein-'
0.63 i- 0.01 1.96 0.10 2.53 & 0.14 2.85 k 0.13 16.5 k 1.2 96.0 k 10 6.91 k 0.37 2.15 f 0.06 0.092 i 0.007
+
* **
DISCUSSION The kinetic analysis of the reaction 2-Oxoglutaramate + phenylalanine Glutamine phenylpyruvate
+
5 1 10 20 20 20 20 20 20 20 20 1 10 10
Table 4. Kinetic parametersf o r the t~un.~amination oj'Z-o.~oat.id.7 [U-'4C]Glutamine (10 mM) was used as the amino group donor except for 2-oxoglutaramate, where phenylalanine (1 mM) was used. Values standard deviation are shown
Glyoxalate 2-0x0-4-methiolbutyrate 3-Mercaptopyruvate Pyruvate 2-Oxobutyrate 2-Oxovalerate 2-Oxoglutaramate P henylpyruvate
K,
V
mM
pmol h - ' mgprotein
1.46 k 0.06 0.25 & 0.01 0.61 k 0.41 13.5 & 0.8 9.19 k 0.48 0.52 6.76 20.5 0.9 0.023 k 0.001
300 k 456 k 239 84+ 108 98k 87+ I76 &
+ +
* *
'
8 15 14 4 5 7 3 7
+
165 3 217 & 11 12 220 167 & 7 64 4 7 63 63 & 3 86 k 2 97 i 4
was studied, and the kinetic parameters calculated from these date are presented in Table 4.
+
100 146 235 89 71 28 37 42 12 8 32 33 0 18 18 7
-
Table 2. Kinetic parameters .for the trunsamination of' umino acids The amino acceptor was phenylpyruvate (0.36 mM) except for L-phenylalanine, where 2-oxoglutaramate (100 mM) was used. With each value the standard deviation is shown. The disappearance or the formation of the end-borate complex of phenylpyruvate was measured
r-Glutamine L-Methionine L-Ethionine 1.-S-Methylcysteine ~-Glutamate-5-methylester ~-Glutamate-5-ethylester ~.-5-N-MethyIglutamine L-Methionine (SR)sulfoxide L-Phenylalanine
mM 1 10
-
0 x 0 acid
Amino acid
Concentration Relative rate with glutamine
is consistent with a ping-pong reaction mechanism, as can be expected for a transamination reaction. The kinetic parameters, K , and V , were calculated according the equation 1 1 v v a h with K, and Kh representing the Michaelis constants of both substrates, and a and b their concentration. When these concentrations are kept at a constant ratio (e.g. h = 100 a),the equation becomes:
The plot (Fig. 3) is in accordance with this equation, as is demonstrated by the excellent agreement of the
F. Van Leuven : Brain Glutamine Transaminase
274
values for the maximal reaction rate obtained in this and the first experiment. The Michaelis constant for 2-oxoglutaramate is rather high, but it should be remembered that this 0x0 acid undergoes spontaneous cyclization. The concentration of the open form, which is probably the reactive molecule in the transamination reaction, amounts to only 0.3'j/, of the total concentration in solution (at neutral pH and 30 "C) [8]. Assuming the same value under the conditions of our experiments, a value of 0.06 mM for the K, of 2-oxoglutaramate is obtained. Although this value may only be an approximation, it probably better reflects the real value for the Michaelis constant of this 0x0 acid. This reasoning holds, provided the interconversion of the cyclic and open chain isomers of 2-0x0glutaramate is not rate-limiting relative to the transamination reaction, which is the case at the high pH in our experiments [8]. The specificity of glutamine transaminase was determined with 30 amino acids. Of the natural amino acids, methionine proved to be a better substrate than glutamine itself. Phenylalanine and tyrosine were also active in the transamination with glyoxalate, as were cysteine and histidine, but at a much slower rate. The glutamine analogues, 5-N-methylglutamine and 5-N-hydroxyglutamine, and the 5-methyl and 5-ethyl esters of glutamic acid, are also substrates for the purified enzyme. The analogues of methionine are even better substrates. That methionine is probably transaminated by the same enzyme as glutamine is indicated by the fact that the maximal velocity of the reaction of glutamine with phenylpyruvate is not changed by the addition of methionine (Fig. 4). The kinetic constants, determined for the most active amino acids, show that the affinity of the enzyme for glutamine is greater than for methionine (Table 2). Considering the fact that the concentration of glutamine in the brain of the rat is much higher than that of methionine, we may speculate that in vivo glutamine is the normal substrate of the enzyme. Of the 0x0 acids that were studied, all showed some activity with the enzyme. The most important were phenylpyruvate, glyoxalate and the 0x0 analogues of tyrosine, methionhe, cysteine, and glutamine. These data indicate that some 0x0 acids are substrates for glutamine transaminase, while the corresponding amino acids are not (at least in our experimental conditions). This means that the enzyme is more specific for the amino acids than for the 0x0 acid substrate. which was also observed for the
0' 0
I
1
I
I
I
2 3 4 [Glutarnine]-' (rnM-')
I
5
Fig.4. Influence of the concrwtrarion of glutaminr on the initiul reaction rule with phenylpyruvate and the effict of methionine. The initial rate was determined with glutamine alone ( 0 ) and with The concentration glutamine plus methionine (1.25 mM) added (0). of phenylpyruvate was measured to determine initial reaction rates
enzyme obtained from sources other than brain [9, lo]. This phenomenon can not be readily explained. When the K,,, values of glutamine, phenylalanine and methionine are compared with those for the corresponding 0x0 analogues, one observes that the enzyme has a greater affinity for the 0x0 acids than for the amino acids. This can be important in that the 0x0 acids will more readily be bound by the enzyme. Although it is an intriguing observation, it is clear that the less stringent specificity of the enzyme for the 0x0 acid substrate is important in vivo. Many 0x0 acids can be aminated by this reaction, with the formation of the corresponding amino acids. Moreover, the excess of ammonia toxic for the brain and stored in glutamine is used in the synthesis of several important compounds.
REFERENCES 1. Van Leuven, F. (1975) Eur. J . Biochem. 58, 153-158. 2. Van Leuven, F. (1974) Arch. Int. Physiol. Biochem. 82, 219283. 3. Meister, A. (1953) .J. Bid. Chcm. 200, 571 -589. 4. Parrod, J . (1942) C. R. Hc>hd.SPances Acad. Sci. (Puri,s) 251. 146- 148. 64, 1021 - 1022. 5. Lichtenstein, N. (1942) J . Am. C'hem. SOC. 6 . Velick, S. F. & Vavra. J. (1962) J . B i d . Chem. 237,2109- 2122. I. Cleland, W . W. (1963) Biochim. Bioph.vs. Acta, 67, 104- 137. 8. Hersh, L. B. (1971) Biochemistry, 10, 2884-2891. 9. Cooper, A. J. L. & Meister, A. (1972) Biochemistry, 11, 661 671. 10. Cooper, A. J. L. & Meister, A. (1974) J . Bid. Chem. 240, 2554- 2561.
F. Van Leuven, Laboratoriuin voor Normale en Pathologische Fysiologie, Fakulteit voor Geneeskunde, Rijksuniversiteit te Gent, De Pintelaan 135, 8-9000 Gent, Belgium
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