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[26] Amino acid reductases
[26]
AMINO ACID REDUCTASES
217
Properties The optimum pH for racemization has been reported at pH 6.81~ and pH 8.0.13 Pyridoxal phosphate stimulates the rate of racemization by dried cells threefold. ~ Glutamine does not stimulate the rate of racemization. ~3 A purified alanine racemase from Streptococcus faecalis does not racemize glutamate. 1~
[26] A m i n o Acid R e d u c t a s e s By R. M. JOHNSTONE and J. H. QUASTEL Stickland, ~ in his studies of amino acid interactions, described the existence of an enzyme in Cl. sporogenes concerned with the reduction of a-amino acids. He demonstrated that glycine, proline, and hydroxyproline are capable of oxidizing reduced benzylviologen in the presence of washed suspensions of the organism. In amino acid interactions, glycine is reduced to acetic acid and ammonia, and proline to 8-aminovaleric acid. Woods 2 found, in addition, with Cl. sporogenes, that ornithine and arginine are both activated as hydrogen acceptors, ornithine giving rise to 8-aminovaleric acid.
Assay Methods Manometric. (1) When an amino acid is used as hydrogen donor, an interaction occurs between the donor and acceptor amino acids, with the evolution of carbon dioxide from the donor. The rate of carbon dioxide evolution is a measure of the reductase activity. 1,3 The following is a typical reaction: CH3CHNH~COOH nu 2CH2NH~.COOH + 2H20 = 3CHsCOOH + 3NH3 ~- CO~ (2) Molecular hydrogen is absorbed by Cl. sporogenes in the presence of amino acid hydrogen acceptors but not of amino acid hydrogen donators. 3,4 The rate of hydrogen absorption may be made a measure of reductase activity. Colorimetric. The amino acid hydrogen acceptors readily oxidize a variety of leuco dyes. The rate of oxidation of reduced benzylviologen 1L. H. Stickland, Biochem. J. 28, 1746 (1934); 29, 288,889 (1935). D. D. Woods, Biochem. J. 30, 1934 (1936). 3 R. NIamelak and J. H. Quastel, Biochim. el Biophys. Acta 12, 103 (1953). 4 j. C. Hoogerheide and W. Kocholaty, Biochem. J. 32, 949 (1938).
218
ENZYMES OF PROTEIN METABOLISM
[26]
has been used successfully by several workers as a measure of amino acid reductase activity. 1,2 Stickland ~ found that glycine, in the presence of Cl. sporogenes, oxidizes leucomethylviologen completely, leucobenzylviologen partly, and leucophenosafranine not at all. Proline oxidizes a variety of leuco dyes including leucophenosafranine. Ammonia Formation. When glycine, ornithine, or arginine is reduced by a hydrogen donor in the presence of Cl. sporogenes ammonia is formed. The rate of ammonia production gives a measure of reductase activity. It should be noted that, when ornithine, arginine, or glycine is used as a hydrogen acceptor for coupled anaerobic amino acid interactions, more ammonia is produced than when proline is used as the hydrogen acceptor. This is due to the fact that proline is not deaminated on reduction in contrast to the other amino acids. Occurrence. Apart from Cl. sporogenes, the following organisms are known to bring about amino acid interactions: 5 C1. butyricum, C1. flabelliferum, Cl. histolyticum, C1. saproticum, Cl. sordelii, C1. bifermentans, Cl. acetobutylicum, Cl. indolicus, and Cl. botulinum.
Preparations Amino acid reductase may be found in resting cell suspensions, lyophilized cells, and cell-free extracts of Cl. sporogenes. Cells grown on a variety of media show good amino acid reductase activities. ~-4,8 Resting Cell Suspensions. The organisms are grown on a suitable medium 2-4,6 for 12 to 16 hours at 37 °. The cells are then centrifuged and washed twice in an isotonic sodium or potassium chloride solution. The packed, washed cells from a liter of medium are suspended in 16 ml. of the same salt solution. One milliliter of this suspension will evolve 300 to 400 ~1. of carbon dioxide per hour at 37 ° from a mixture of 0.02 M alanine and 0.02 M proline in a 0.028 M bicarbonate medium in an atmosphere of 93% N2 and 7% C02. The optimum pH for amino acid reductases is 7.2 to 7.4. When molecular hydrogen is used as hydrogen donor, it is found advisable to boil salines and buffer solution preparatory to use 4 in order to remove oxygen, as hydrogenase is easily inactivated by air. Johnstone and Quastel (unpublished results) find that the presence of 0.1% neutral cysteine (or sodium pyruvate) in the wash saline will prevent loss of hydrogenase activity during the preparation of the resting cells. Care must be taken to avoid unnecessary exposure to air in the course of preparation. B. Nisman, M. Raynaud, and G. H. Cohen, Ann. inst. Pasteur 74, 323 (1948). e B. Nisman and G. Vinet, Ann. inst. Pasteur 78, 115 (1950).
[26]
AMINO ACID REDUCTASES
219
Preparation of Lyophilized Cells. 8 Packed cells of C1. sporogenes, prepared as above, are washed twice with 1% sodium thioglycoUate solution and suspended in 20 ml. of 0.15 M KC1. The cell suspension is frozen in a mixture of dry ice and acetone and lyophilized for 5 hours. The dried cells are stored in a vacuum desiccator at 0 to 10 °. For assay, 18 to 20 mg. of the dried cells is used per milliliter. Such a preparation shows considerable prolinc and ornithine reductase activities, b u t the activity for glycine is absent. Preparation of Bacterial Extracts. A bacterial press described by Hughes 7 was used b y Mamelak and Quastel ~ for preparation of cell-free extracts of Cl. sporogenes. The cells from 2 1. of medium, grown as described earlier, are harvested, washed once with 200 ml. of 1% sodium thioglycollate solution, and a second time with 10 ml. of this solution. T h e packed, washed cells are frozen for 5 to 10 minutes in dry ice and inserted into the press which has previously been cooled to - 2 0 ° . The crushed cells are suspended in 7 to 9 ml. of 1% sodium thioglycollate solution and centrifuged for 2 minutes at 20,000 X g at 0 °. T h e amber-colored supernatant fluid reduces proline or ornithine in the presence of suitable hydrogen donors 3 but will not reduce glycine (unpublished result). Properties
Stability. Exposure of a resting cell suspension of Cl. sporogenes to air for 3 or 4 hours causes a marked fall in the rates of the anaerobic amino acid interactions. 3 This fall is due to the presence of oxygen, as no diminution is experienced if the suspension is exposed to nitrogen for the same length of time. Exposure to small concentrations of hydrogen peroxide (1/50,000) for a few minutes is equally inhibitory. Reductase activity may be maintained for more than 2 weeks if the packed cells are stored at - 2 0 ° (unpublished result). If the cells are kept in suspension at 0 to 20 °, the activity is completely lost in less than 6 hours. Specificity. Woods 2 showed that either optical isomer of proline or ornithine is reduced b y a suitable hydrogen donor in the presence of C1. sporogenes. He ~ also demonstrated t h a t the amino group of glycine is essential for reductase activity, since replacement of the amino group b y a hydroxyl group results in a complete loss of the ability of the molecule to act as a hydrogen acceptor. N - M e t h y l glycine, N-acetyl glycine, and ethanolamine are not attacked b y the enzyme, nor do they inhibit glycine reductase (Johnstone and Quastel, unpublished results). D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951).
220
EIgZYMES OF PROTEIN METABOLISM
[27]
Little is known at present of the specificity of amino acid reductases. The fact that the ability of Cl. sporogenes extracts to reduce glycine is lost under conditions where proline and ornithine reductions may still take place indicates the likelihood that separate enzymes are involved in the reduction of these amino acids. Necessity for DPN. Recent work by Mamelak and Quastel ~ has shown that the coenzyme involved in the reduction of proline and ornithine is DPN. It would appear, therefore, that amino acid reductase of C1. sporogenes is DPN-linked. Inhibitors. Stickland ~ demonstrated that arsenite inhibits amino acid interactions in C1. sporogenes. Nisman and Vinet Bshowed, however, that the oxidation of the amino acid donor is unaffected by arsenite (M/225), whereas the reductions of proline and glycine are completely suppressed. h/Iamelak and Quastel 3 demonstrated that organic arsenoxides, particularly m-amino-p-hydroxyphenylarsenoxide, are inhibitory to amino acid interactions in C1. sporogenes. They showed that the reduction of proline is affected more than the oxidation of alanine. For example, 8 "/ of the above organic arsenoxide per milliliter caused a 50% inhibition of the amino acid interaction but had no effect on the aerobic oxidation of alanine. ~ Pentavalent arsenic compounds have little or no toxicity. The inhibitions caused by organic arsenoxides can be largely reversed by the presence of thiol compounds such as glutathione2 The evidence indicates that the amino acid reductases are labile thiol enzymes.
[27] L - G l u t a m i c D e h y d r o g e n a s e
from Liver
L-Glutamate- ~- D P N + ~ H:O ~ ~-Ketoglutarate--~ D P N H ~ NH4 + ~- H +
By HAROLD J. STRECKER Assay Method Principle. The oxidation of glutamate is measured by the increase of optical density at 340 m~ caused by the reduction of DPN. Alternately the reductive amination of a-ketoglutarate may be measured by the decrease in optical density arising from the oxidation of D P N H . Reagents Potassium glutamate (0.5 M). 918 mg. of glutamie acid hydrochloride is suspended in 5 ml. of H~O. Sufficient K O H solution is
AMINO ACID REDUCTASES
217
Properties The optimum pH for racemization has been reported at pH 6.81~ and pH 8.0.13 Pyridoxal phosphate stimulates the rate of racemization by dried cells threefold. ~ Glutamine does not stimulate the rate of racemization. ~3 A purified alanine racemase from Streptococcus faecalis does not racemize glutamate. 1~
[26] A m i n o Acid R e d u c t a s e s By R. M. JOHNSTONE and J. H. QUASTEL Stickland, ~ in his studies of amino acid interactions, described the existence of an enzyme in Cl. sporogenes concerned with the reduction of a-amino acids. He demonstrated that glycine, proline, and hydroxyproline are capable of oxidizing reduced benzylviologen in the presence of washed suspensions of the organism. In amino acid interactions, glycine is reduced to acetic acid and ammonia, and proline to 8-aminovaleric acid. Woods 2 found, in addition, with Cl. sporogenes, that ornithine and arginine are both activated as hydrogen acceptors, ornithine giving rise to 8-aminovaleric acid.
Assay Methods Manometric. (1) When an amino acid is used as hydrogen donor, an interaction occurs between the donor and acceptor amino acids, with the evolution of carbon dioxide from the donor. The rate of carbon dioxide evolution is a measure of the reductase activity. 1,3 The following is a typical reaction: CH3CHNH~COOH nu 2CH2NH~.COOH + 2H20 = 3CHsCOOH + 3NH3 ~- CO~ (2) Molecular hydrogen is absorbed by Cl. sporogenes in the presence of amino acid hydrogen acceptors but not of amino acid hydrogen donators. 3,4 The rate of hydrogen absorption may be made a measure of reductase activity. Colorimetric. The amino acid hydrogen acceptors readily oxidize a variety of leuco dyes. The rate of oxidation of reduced benzylviologen 1L. H. Stickland, Biochem. J. 28, 1746 (1934); 29, 288,889 (1935). D. D. Woods, Biochem. J. 30, 1934 (1936). 3 R. NIamelak and J. H. Quastel, Biochim. el Biophys. Acta 12, 103 (1953). 4 j. C. Hoogerheide and W. Kocholaty, Biochem. J. 32, 949 (1938).
218
ENZYMES OF PROTEIN METABOLISM
[26]
has been used successfully by several workers as a measure of amino acid reductase activity. 1,2 Stickland ~ found that glycine, in the presence of Cl. sporogenes, oxidizes leucomethylviologen completely, leucobenzylviologen partly, and leucophenosafranine not at all. Proline oxidizes a variety of leuco dyes including leucophenosafranine. Ammonia Formation. When glycine, ornithine, or arginine is reduced by a hydrogen donor in the presence of Cl. sporogenes ammonia is formed. The rate of ammonia production gives a measure of reductase activity. It should be noted that, when ornithine, arginine, or glycine is used as a hydrogen acceptor for coupled anaerobic amino acid interactions, more ammonia is produced than when proline is used as the hydrogen acceptor. This is due to the fact that proline is not deaminated on reduction in contrast to the other amino acids. Occurrence. Apart from Cl. sporogenes, the following organisms are known to bring about amino acid interactions: 5 C1. butyricum, C1. flabelliferum, Cl. histolyticum, C1. saproticum, Cl. sordelii, C1. bifermentans, Cl. acetobutylicum, Cl. indolicus, and Cl. botulinum.
Preparations Amino acid reductase may be found in resting cell suspensions, lyophilized cells, and cell-free extracts of Cl. sporogenes. Cells grown on a variety of media show good amino acid reductase activities. ~-4,8 Resting Cell Suspensions. The organisms are grown on a suitable medium 2-4,6 for 12 to 16 hours at 37 °. The cells are then centrifuged and washed twice in an isotonic sodium or potassium chloride solution. The packed, washed cells from a liter of medium are suspended in 16 ml. of the same salt solution. One milliliter of this suspension will evolve 300 to 400 ~1. of carbon dioxide per hour at 37 ° from a mixture of 0.02 M alanine and 0.02 M proline in a 0.028 M bicarbonate medium in an atmosphere of 93% N2 and 7% C02. The optimum pH for amino acid reductases is 7.2 to 7.4. When molecular hydrogen is used as hydrogen donor, it is found advisable to boil salines and buffer solution preparatory to use 4 in order to remove oxygen, as hydrogenase is easily inactivated by air. Johnstone and Quastel (unpublished results) find that the presence of 0.1% neutral cysteine (or sodium pyruvate) in the wash saline will prevent loss of hydrogenase activity during the preparation of the resting cells. Care must be taken to avoid unnecessary exposure to air in the course of preparation. B. Nisman, M. Raynaud, and G. H. Cohen, Ann. inst. Pasteur 74, 323 (1948). e B. Nisman and G. Vinet, Ann. inst. Pasteur 78, 115 (1950).
[26]
AMINO ACID REDUCTASES
219
Preparation of Lyophilized Cells. 8 Packed cells of C1. sporogenes, prepared as above, are washed twice with 1% sodium thioglycoUate solution and suspended in 20 ml. of 0.15 M KC1. The cell suspension is frozen in a mixture of dry ice and acetone and lyophilized for 5 hours. The dried cells are stored in a vacuum desiccator at 0 to 10 °. For assay, 18 to 20 mg. of the dried cells is used per milliliter. Such a preparation shows considerable prolinc and ornithine reductase activities, b u t the activity for glycine is absent. Preparation of Bacterial Extracts. A bacterial press described by Hughes 7 was used b y Mamelak and Quastel ~ for preparation of cell-free extracts of Cl. sporogenes. The cells from 2 1. of medium, grown as described earlier, are harvested, washed once with 200 ml. of 1% sodium thioglycollate solution, and a second time with 10 ml. of this solution. T h e packed, washed cells are frozen for 5 to 10 minutes in dry ice and inserted into the press which has previously been cooled to - 2 0 ° . The crushed cells are suspended in 7 to 9 ml. of 1% sodium thioglycollate solution and centrifuged for 2 minutes at 20,000 X g at 0 °. T h e amber-colored supernatant fluid reduces proline or ornithine in the presence of suitable hydrogen donors 3 but will not reduce glycine (unpublished result). Properties
Stability. Exposure of a resting cell suspension of Cl. sporogenes to air for 3 or 4 hours causes a marked fall in the rates of the anaerobic amino acid interactions. 3 This fall is due to the presence of oxygen, as no diminution is experienced if the suspension is exposed to nitrogen for the same length of time. Exposure to small concentrations of hydrogen peroxide (1/50,000) for a few minutes is equally inhibitory. Reductase activity may be maintained for more than 2 weeks if the packed cells are stored at - 2 0 ° (unpublished result). If the cells are kept in suspension at 0 to 20 °, the activity is completely lost in less than 6 hours. Specificity. Woods 2 showed that either optical isomer of proline or ornithine is reduced b y a suitable hydrogen donor in the presence of C1. sporogenes. He ~ also demonstrated t h a t the amino group of glycine is essential for reductase activity, since replacement of the amino group b y a hydroxyl group results in a complete loss of the ability of the molecule to act as a hydrogen acceptor. N - M e t h y l glycine, N-acetyl glycine, and ethanolamine are not attacked b y the enzyme, nor do they inhibit glycine reductase (Johnstone and Quastel, unpublished results). D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951).
220
EIgZYMES OF PROTEIN METABOLISM
[27]
Little is known at present of the specificity of amino acid reductases. The fact that the ability of Cl. sporogenes extracts to reduce glycine is lost under conditions where proline and ornithine reductions may still take place indicates the likelihood that separate enzymes are involved in the reduction of these amino acids. Necessity for DPN. Recent work by Mamelak and Quastel ~ has shown that the coenzyme involved in the reduction of proline and ornithine is DPN. It would appear, therefore, that amino acid reductase of C1. sporogenes is DPN-linked. Inhibitors. Stickland ~ demonstrated that arsenite inhibits amino acid interactions in C1. sporogenes. Nisman and Vinet Bshowed, however, that the oxidation of the amino acid donor is unaffected by arsenite (M/225), whereas the reductions of proline and glycine are completely suppressed. h/Iamelak and Quastel 3 demonstrated that organic arsenoxides, particularly m-amino-p-hydroxyphenylarsenoxide, are inhibitory to amino acid interactions in C1. sporogenes. They showed that the reduction of proline is affected more than the oxidation of alanine. For example, 8 "/ of the above organic arsenoxide per milliliter caused a 50% inhibition of the amino acid interaction but had no effect on the aerobic oxidation of alanine. ~ Pentavalent arsenic compounds have little or no toxicity. The inhibitions caused by organic arsenoxides can be largely reversed by the presence of thiol compounds such as glutathione2 The evidence indicates that the amino acid reductases are labile thiol enzymes.
[27] L - G l u t a m i c D e h y d r o g e n a s e
from Liver
L-Glutamate- ~- D P N + ~ H:O ~ ~-Ketoglutarate--~ D P N H ~ NH4 + ~- H +
By HAROLD J. STRECKER Assay Method Principle. The oxidation of glutamate is measured by the increase of optical density at 340 m~ caused by the reduction of DPN. Alternately the reductive amination of a-ketoglutarate may be measured by the decrease in optical density arising from the oxidation of D P N H . Reagents Potassium glutamate (0.5 M). 918 mg. of glutamie acid hydrochloride is suspended in 5 ml. of H~O. Sufficient K O H solution is