- Email: [email protected]
[83] Kynurenine aminotransferase from kidney supernatant
664
a-AMINOADIPATE
[83]
The steps in the formation of cephalosporin C from penicillin N have been deduced mainly through the use of blocked mutants. 120 Patterns of accumulation of intermediates are consistent with a biosynthetic sequential conversion of penicillin N to cephalosporin C through deacetoxycephalosporin C and deacetylcephalosporin C. Conversion of penicillin N to deactoxycephalosporin C is an oxidative expansion from a five-membered to a six-membered ring. The mechanism of this expansion is not known. The cephamycin antibiotics are formed by enzymatic transformations of deacetylcephalosporin C. 8
Enzymes The enzyme reported for which a-aminoadipate is a substrate or product of catalysis include a-ketoadipate : glutamate aminotransferase from bakers' yeast and Neurospora, 51 a-aminoadipate reductase from bakers' yeast, 51 a-aminoadipate-8-semialdehyde : NAD oxidoreductase. 68,69 and a-aminoadipate transaminase from P. putida, 7° and a-aminoadipate : aketoglutarate aminotransferase from rat liver 1°6 and kidney. 106,~07,~40 ~40See this volume [831.
[83] K y n u r e n i n e A m i n o t r a n s f e r a s e f r o m K i d n e y S u p e r n a t a n t By RICHARD A. HARTLINE +
N H3
o
-OOC-ICH21a-CH-COO-
<
~-
Glutamate ~
Oil > -OOC-ICH213-C-COO~ Ketoadipate
*< - Aminoadipate
The e-kynurenine:a-ketoglutarate aminotransferase (cyclizing) (EC 2.6.1.7) activity of rat kidney supernatant, an enzyme in tryptophan catabolism, copurifies with the activity of a-aminoadipate : a-ketoglutarate aminotransferase. 1 Evidence that the two activities are properties of a single protein are (1) copurification of the two activities with the ratio of their specific activities remaining constant, (2) similar chromatographic I M. C. Tobes
and M. Mason,
J. Biol. Chem. 2 5 2 , 4591 ( 1 9 7 7 ) .
METHODS IN ENZYMOLOGY, VOL. 113
Copyright ~t~ 1985 by Academic Press, Inc. All righls of reproduction in any form reserved.
[83]
KYNURENINE AMINOTRANSFERASE
o .'.~
~
C-CH2-C-COO-
665
o_.~ o~-Ket0glutarate Glutamate
-NH2
COO-
L-Kynurenine
Kynurenate
T
I N÷H3
°<-Ket°glutarate
Glutamate H O~- ~ _ , / C H 2 -
0C-COO-
T
T
L- 3, 5-Diiodotyrosine
3, 5-Diiodo-4-hydroxyphenylpyruvate
FIG. 1. Reactions catalyzed by the kynurenine and halogenated tyrosine aminotrans-
ferase activities of mammalian a-aminoadipate (kynurenine):a-ketoglutarate ferase.
aminotrans-
behavior, (3) similarity in their dependence on added pyridoxal-5'-phosphate for activity, and (4) similar patterns of heat inactivation. It has been suggested that the presence of aminoadipate (kynurenine) aminotransferase in kidney may represent an enzyme associated with lysine catabolism rather than with tryptophan degradation. ~,2 Consistent with this concept is the recent demonstration of lysine : a-ketoglutarate reductase and saccharopine dehydrogenase activities in human kidney. 3 A role in lysine catabolism could justify the presence of the relatively high level of kynurenine aminotransferase activity in kidney 4,5 which lacks a complete pathway for tryptophan catabolism, a-Aminoadipate (kynurenine) aminotransferase also catalyzes aminotransfer of 3,5-diiodotyrosine (halogenated tyrosine aminotransferase) suggesting a possible role in thyroid hormone metabolism. 6 The reactions catalyzed by the kynurenine and halogenated tyrosine aminotransferase activities are illustrated in Fig. 1. ~-Aminoadipate (kynurenine):o~-ketoglutarate aminotransferase activity is also present in rat kidney mitochondria 6 and the supernatant 7 and 2 B. D. Manning and M. Mason, Life Sci. 17, 225 (1975). 3 j. Hutzler and J. Dancis, Biochim. Biophys. Acta 377, 42 (1975). 4 M. M a s o n , J. Biol. Chem. 211, 839 (1954). 5 M. Mason and C. P. Berg, J. Biol. Chem. 188, 783 (1951). 6 M. C. Tobes and M. Mason, Life Sci. 22, 793 (1978). 7 M. Tobes and M. Mason, Biochern. Biophys. Res. Commun. 62, 390 (1975).
666
O~-AMINOADIPATE
[83]
mitochondrial8 fractions of rat liver. The kidney6 and livers mitochondrial enzymes have been partially purified. L-a-Aminoadipate Aminotransferase Assay a
L - o< - A m i n o a d i p a t e
+ o<-Ketoglutarate
~
~ o<-Ketoadipate + G l u t a m a t e b
Activity of this enzyme can be measured from direction a or b by the rate of disappearance (direction a) or appearance (direction b) of a-ketoglutarate with glutamate dehydrogenase. 8 Reagents
Potassium phosphate, pH 7.5 Pyridoxal-5'-phosphate a-Ketoglutarate, pH 7.5 Potassium L-a-aminoadipate, 0.1 M, pH 7.5 HC1, 1 M KOH, 1 M Ammonium chloride NADH Glutamate dehydrogenase Procedure in the " a " Direction. The assay mixture contains 167 mM potassium phosphate buffer (pH 7.5), 0.26 mM pyridoxal-5'-phosphate, 1.67 mM a-ketoglutarate, and enzyme in a volume of 0.3 ml. After a 5 min incubation at 37°, the reaction is initiated by addition of 0.2 ml of 0.1 M potassium L-a-aminoadipate and incubated for 10 min at 37°. Incubation is terminated by addition of 0.1 ml 1 M HCI and the reaction mixture neutralized with 0.1 ml 1 M KOH. A 0.3-ml aliquot is analyzed for a-ketoglutarate. A unit of enzyme activity is defined as the amount of enzyme producing the disappearance of 1/zmol of a-ketoglutarate per min. The aketoglutarate that disappears is calculated by subtracting the a-ketoglutarate remaining from the a-ketoglutarate in a control containing no enzyme. Procedure in the " b " Direction. Assay conditions are the same as for the " a " direction except that a-ketoglutarate and L-a-aminoadipate were replaced by a-ketoadipate and L-glutamate. With this assay the reaction is linear with respect to time and enzyme concentration until 0.1/xmol of aketoglutarate is produced. A unit of enzyme activity is defined as the amount of enzyme producing 1 /xmol of a-ketoglutarate per min. 8 y. Nakatani, M. Fujioka, and K. Higashino,Biochim. Biophys. Acta 198, 219 (1970).
[83]
KYNURENINEAMINOTRANSFERASE
667
Tobes and Mason 7 report the use of this assay with some modifications which include (1) potassium phosphate buffer at pH 7.0 rather than 7.5, (2) neutralization after addition of 0.1 ml 1 M HC1 with 0.1 ml 1.1 M KOH to pH 7.5 rather than I M KOH to pH 7.0, and (3) centrifugation after neutralization at 5000 g for 5 min to remove precipitates. They also found that transferase activity in a mitochondrial suspension is increased by addition of CaC12 to 2 mM. The CaCI2 has no effect on glutamate dehydrogenase activity. Addition of CaCI2 does not increase transferase activity in the supernatant fraction of disrupted mitochondria. ct-Ketoglutarate Determination. a-Ketoglutarate is determined by the amount of N A D H oxidized in the presence of NH4 + and glutamate dehydrogenase according to the following equation: NADH * H + o<-Ketoglutarate 4- NH+4
~
NAD+ -~
> Glutamate
An incubation mixture contains 100 mM potassium phosphate buffer (pH 7.5), 50 mM NH4C1, 0.1 mM NADH, and 0.3 ml of neutralized aminotransferase reaction mixture in a final volume of 3.0 ml. The reaction is initiated by addition of glutamate dehydrogenase and the oxidation of N A D H followed spectrophotometrically at 340 nm. The molar absorptivity of N A D H at 340 nm is 6220. A unit of enzyme activity is defined as the amount of enzyme oxidizing 1 ~mol of NADH per min. Kynurenine Aminotransferase Assay L-Kynurenine + o< -Ketoglutarate
) Kynurenate + G l u t a m a t e
The activity of kynurenine aminotransferase is measured spectrophotometrically by following the appearance of kynurenate. 7
Reagents Pyridoxal-5'-phosphate a-Ketoglutarate, pH 6.5 Imidazole-HCi, pH 6.5 e-Kynurenine sulfate, pH 6.5 1% Boric acid in 95% ethanol Procedure. The incubation mixture contains 0.20 mM pyridoxal-5'phosphate, 5.00 mM a-ketoglutarate, 3.26 mM L-kynurenine sulfate, 100 mM imidazole-HCi buffer (pH 6.5), and 0 to 0.1 ml of enzyme solution in a final volume of 0.60 ml. The mixture is incubated at room temperature for 10 min before the reaction is initiated by addition of L-kynurenine sulfate. After a 30 min incubation at 37° the reaction is terminated by addition of
668
a-AMINOADIPATE
[83]
10 ml of ethanolic boric acid solution. Precipitates are removed by centrifugation at 5000 g for 5 min. Kynurenate formed is determined spectrophotometrically at 333 nm with correction for any kynurenine present by its absorption at 365 nm. Kynurenate absorbance is calculated according to the equation9: Absorbance at 333 nm due to kynurenate = (absorbance at 333 nm) - (0.39 x absorbance at 365 nm) The molar absorptivity of kynurenate at 333 nm is 8850.~° A unit of enzyme activity is defined as the amount of enzyme producing 1 /~mol of kynurenate per min. 3,5-Diiodotyrosine Aminotransferase Assay L - 3, 5 - D i i o d o t y r o s i n e + o< - K e t o g l u t a r a t e
> L-3,5
-Diiodo-4-hydroxyphenylpyruvate
+ Glutamat~
This transferase activity is determined by following the formation of the 3,5-diiodo-4-hydroxyphenylpyruvate product measured spectrophotometrically as the enol-borate complex.~l
Reagents Pyridoxal-5'-phosphate L-3,5-Diiodotyrosine, pH 7.0 or 6.5 Sodium phosphate, pH 7.0 or 6.5 a-Ketoglutarate, pH 7.0 or 6.5 H2SO4, 6 N Arsenate, 2 M, pH 6.5 Arsenate, 2 M which is I M in borate, pH 6.5
Procedure. The reaction mixture contains the 25 gM pyridoxal-5'phosphate, 2.5 mM L-3,5-diiodotyrosine, and 31.3 mM sodium phosphate buffer which is 0.31 mM in EDTA at pH 7.0 jl or 6.5, 6 and the enzyme in a final volume of 1.6 ml. After a 5 min incubation period at 37° the reaction is initiated by addition of a-ketoglutarate to 16 mM. The reaction is stopped, after incubation for 10 to 210 min at 37°, by addition of 0.1 ml of 6 N H2SO4. A 0.7-ml aliquot of the acidified incubation mixture is added to 2 ml of the arsenate solution (control). A second 0.7-ml aliquot is added to 2 ml of the arsenate-borate solution (enol-borate sample). Absorbance of the enol-borate sample is read at 330 nm. The molar absorptivity of the enol-borate complex of 3,5-diiodo-4-hydroxyphenylpyruvate at 330 nm is 9 M. Tobes, personal c o m m u n i c a t i o n . z0 W. E. K n o x , Biochem. J. 53, 379 (1953). H M. N a k a n o , J. Biol. Chem. 242, 73 (1967).
[83]
KYNURENINE AMINOTRANSFERASE
669
8110. H A unit of activity is defined as the amount of enzyme required for the formation of 1/zmol of 3,5-diiodo-4-hydroxyphenylpyruvateper min. Purification ~ All procedures are carried out at 0 to 4°. Solutions are prepared with redistilled and deionized water. Buffers are all potassium phosphate containing 10 mM 2-mercaptoethanol. Male adult albino rats (250 to 350 g) are sacrificed by decapitation and the kidneys immediately placed on ice. The kidneys are weighed, placed in a cold homogenizer in 4 ml of 0.25 M sucrose per g of tissue, homogenized, and centrifuged at 30,000 g for 20 min. 12,13 The supernatant is decanted and frozen immediately. The frozen supernatant is usually used within 3 weeks but loses little activity over a 3-month period at - 1 5 °. Kidneys from 30 rats yield approximately 200 ml of supernatant. Copurification of o~-aminoadipate and kynurenine aminotransferase activities is summarized in the table. Step I. A c i d Precipitation. Approximately 200 ml of frozen supernatant is thawed. The solution is made 10/zM in pyridoxal-5'-phosphate, titrated to pH 5.25 with 1.0 M acetic acid, and allowed to stand 5 min with occasional stirring. After centrifugation at 30,000 g for 10 min the supernatant is titrated to pH 6.5 with 1.0 M NaOH. Step 2. A m m o n i u m Sulfate and Heat Fractionation. Supernatant from the acid precipitation step is made 35% saturated with solid ammonium sulfate 14 and, while maintaining the pH at 6.5 with addition of 1.0 M NaOH, the suspension is stirred for 1 hr. The solution is made 0.05 mM in pyridoxal-5'-phosphate, taken to 55° for 5 min with constant stirring, brought back to 4° in an ice bath, and centrifuged at 30,000 g for 20 min. The supernatant is taken to 65% saturation in ammonium sulfate, stirred for 1 to 2 hr while maintaining the pH at 6.5 with 1.0 M NaOH, and the resulting suspension centrifuged at 30,000 g for 20 min. The supernatant is discarded and the precipitate dissolved in a minimal volume (20 to 24 ml) of 8 mM buffer at pH 7.1 and dialyzed against 2.1 liters of buffer for 19 hr with one change of buffer. Step 3. DEAE-Cellulose Chromatography. A 3.4 × 9.0 cm column is equilibrated with 8 mM buffer, pH 7.1, and the dialyzed solution applied to the column at approximately 45 ml/hr. Collecting 5 ml fractions, the enzyme is eluted off with the same buffer at the same flow rate. Aminotransferase activities which appear between fractions 40 to 60 are combined. 12M. Mason, J. Ford, and H. L. C. Wu, Ann. N.Y. Acad. Sci. 166(1), 170 (1969). ~3M. Mason, Biochem. Biophys. Res. Commun. 60, 64 (1974). 14M. Dixonand E. C. Webb, "Enzymes," 2nd ed., p. 40. AcademicPress, New York, 1964.
o
©
z
z
z
<~ z~ z
z
<
,,,
z
m
z
o
~z <
© z <
© Z
_o {.-
r, o
k~
[83]
KYNURENINE AMINOTRANSFERASE
671
Step 4. Hydroxylapatite I Chromatography. A 2.2 x 18.0 cm column is equilibrated with 8 mM buffer at pH 7.0 and the pooled DEAE eluate applied at 25 ml/hr. The column is eluted with 1.5 column volumes of 8 mM buffer and then eluted, at the same flow rate, with a linear gradient of 200 ml of 8 mM buffer, pH 7.0, and 200 ml of 210 mM buffer, pH 7.0. Fractions of 4 ml are collected and the active ones, from 117 to 128, are pooled and concentrated to 10 ml by means of a 50 ml Diaflo cell with a PM-10 membrane at an N2 pressure of 10 to 15 psi. The concentrated enzyme is dialyzed overnight with one change of buffer against 1 liter of 70 mM buffer at pH 7.0. Step 5. Hydroxylapatite H Chromatography. A 1.0 x 30.0 cm column is equilibrated with 70 mM buffer, pH 7.0, and the dialyzed sample applied at the rate of 7 ml/hr. Elution of the column is initiated with 1.5 column volumes of 70 mM buffer, pH 7.0, followed by a linear gradient elution of 94 ml of 70 mM buffer, pH 7.0, and 94 ml of 140 mM buffer at pH 7.0 at 8-9 ml/hr. Fractions of 2 ml are collected. The enzyme activity elutes off from fractions 65 to 75. With one change of buffer, the pooled active fractions are dialyzed overnight against 1 liter of 70 mM buffer, pH 7.0, containing 0.20 mM pyridoxal-5'-phosphate. Step 6. Hydroxylapatite III Chromatography. A 1.0 x 33.0 cm column is equilibrated with 70 mM buffer, pH 7.0, and the dialyzed sample applied at 5 ml/hr. The initial elution is with 1.5 column volumes of 70 mM buffer at pH 7.0 followed by elution of enzyme activity with a linear gradient of 100 ml of 70 mM buffer, pH 7.0, and 100 ml of 140 mM buffer, pH 7.0, at a rate of 4 to 5 ml/hr. Collecting 2 ml fractions, the enzyme activity elutes from fractions 75 to 85. The pooled active fractions are made 0.20 mM in pyridoxal-5'-phosphate and concentrated to 2 ml by ultrafiltration in a 10 ml Diaflo cell with a PM-10 membrane at a N2 pressure of 10 psi. After concentration, the enzyme is stored at 4° in 70 mM buffer, pH 7.0, that is 0.20 mM pyridoxal-5'-phosphate and 10 mM in 2-mercaptoethanol. The protein concentration of the stored enzyme is approximately !.0 mg/ml. After all chromatography steps the a-aminoadipate and kynurenine aminotransferase activities are dependent on added pyridoxal-5'-phosphate. Therefore, the enzyme purified as the apoenzyme. The overall purification with respect to the crude supernatant is 283fold for o~-aminoadipate aminotransferase activity and 294-fold for kynurenine aminotransferase activity. Purification relative to the crude homogenate is 600-fold. The enzyme is nearly homogeneous according to analytical disc gel electrophoresis at pH 8.9 and 7.5, isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
672
O~-AMINOADIPATE
[83]
Stability. The apoenzyme is most stable under the purification conditions in the pH range of 6.5 to 7.5. Maximal activity is dependent on the presence of a reducing agent such as 2-mercaptoethanol. The apoenzyme is quite stable in phosphate buffer, but when dialyzed against Tris-HC1 or imidazole-HC1, the enzyme activities are rapidly and irreversibly lost. In the Tris or imidazole buffers the apoenzyme is stable only in the presence of saturating levels of pyridoxal-5'-phosphate. Physical Characteristics The average molecular weight of the enzyme is 85,000. The average sedimentation coefficient is 5.8. No differences are observed for the sedimentation coefficient for sedimentation of apo- versus holoenzyme and crude versus purified enzyme. The enzyme is composed of two similar sized subunits (45,000 -+ 850) that are not linked by intrachain disulfide bonds. The isoelectric point of the enzyme is 6.56 -+ 0.06. Specificity
The Purified Enzyme from Rat Kidney. a-Aminoadipate is more reactive with the enzyme than kynurenine (see the table). While no data are presented, 1 the specificity with other amino acids, using a-ketoglutarate as the amino-acceptor, is described to be similar to those observed with a partially purified preparation from rat liver mitochondria. 8 The exception to this similarity is activity with aspartate (see the table). While the available data are consistent with activity with aspartate reflecting a slight contamination by aspartate aminotransferase, the possibility remains that this activity is intrinsic. Additional specificities reported are strong activity with 3,5-diiodotyrosine 6 and weak activities with tryptophan, phenylalanine, and tyrosine.15 The Partially Purified Enzyme from Rat Liver. The specificity of a partially purified preparation of a-aminoadipate : a-ketoglutarate aminotransferase from rat liver mitochondria has been reported. 8 On a relative activity scale in which L-a-aminoadipate is assigned an arbitrary value of 100 and in which a-ketoglutarate is used as the amino-acceptor, OL-aaminopimelate showed 14 and L-norleucine 15. Amino acids that are less than 2 include L-aspartate, L-alanine, L-a-aminobutyrate, glycine, Lvaline, L-leucine, L-isoleucine, L-norvaline, L-e-aminocaproate, Lthreonine, L-serine, L-homoserine, L-cysteine, L-methionine, L-lysine, Larginine, L-citrilline, L-ornithine, L-histidine, L-tyrosine, L-tryptophan, L-phenylalanine, L-proline, and L-glutamate. 15 M. C. Tobes, Ph.D. Thesis, University of Michigan, Ann Arbor (1976).
a-AMINOADIPATE
[83]
The steps in the formation of cephalosporin C from penicillin N have been deduced mainly through the use of blocked mutants. 120 Patterns of accumulation of intermediates are consistent with a biosynthetic sequential conversion of penicillin N to cephalosporin C through deacetoxycephalosporin C and deacetylcephalosporin C. Conversion of penicillin N to deactoxycephalosporin C is an oxidative expansion from a five-membered to a six-membered ring. The mechanism of this expansion is not known. The cephamycin antibiotics are formed by enzymatic transformations of deacetylcephalosporin C. 8
Enzymes The enzyme reported for which a-aminoadipate is a substrate or product of catalysis include a-ketoadipate : glutamate aminotransferase from bakers' yeast and Neurospora, 51 a-aminoadipate reductase from bakers' yeast, 51 a-aminoadipate-8-semialdehyde : NAD oxidoreductase. 68,69 and a-aminoadipate transaminase from P. putida, 7° and a-aminoadipate : aketoglutarate aminotransferase from rat liver 1°6 and kidney. 106,~07,~40 ~40See this volume [831.
[83] K y n u r e n i n e A m i n o t r a n s f e r a s e f r o m K i d n e y S u p e r n a t a n t By RICHARD A. HARTLINE +
N H3
o
-OOC-ICH21a-CH-COO-
<
~-
Glutamate ~
Oil > -OOC-ICH213-C-COO~ Ketoadipate
*< - Aminoadipate
The e-kynurenine:a-ketoglutarate aminotransferase (cyclizing) (EC 2.6.1.7) activity of rat kidney supernatant, an enzyme in tryptophan catabolism, copurifies with the activity of a-aminoadipate : a-ketoglutarate aminotransferase. 1 Evidence that the two activities are properties of a single protein are (1) copurification of the two activities with the ratio of their specific activities remaining constant, (2) similar chromatographic I M. C. Tobes
and M. Mason,
J. Biol. Chem. 2 5 2 , 4591 ( 1 9 7 7 ) .
METHODS IN ENZYMOLOGY, VOL. 113
Copyright ~t~ 1985 by Academic Press, Inc. All righls of reproduction in any form reserved.
[83]
KYNURENINE AMINOTRANSFERASE
o .'.~
~
C-CH2-C-COO-
665
o_.~ o~-Ket0glutarate Glutamate
-NH2
COO-
L-Kynurenine
Kynurenate
T
I N÷H3
°<-Ket°glutarate
Glutamate H O~- ~ _ , / C H 2 -
0C-COO-
T
T
L- 3, 5-Diiodotyrosine
3, 5-Diiodo-4-hydroxyphenylpyruvate
FIG. 1. Reactions catalyzed by the kynurenine and halogenated tyrosine aminotrans-
ferase activities of mammalian a-aminoadipate (kynurenine):a-ketoglutarate ferase.
aminotrans-
behavior, (3) similarity in their dependence on added pyridoxal-5'-phosphate for activity, and (4) similar patterns of heat inactivation. It has been suggested that the presence of aminoadipate (kynurenine) aminotransferase in kidney may represent an enzyme associated with lysine catabolism rather than with tryptophan degradation. ~,2 Consistent with this concept is the recent demonstration of lysine : a-ketoglutarate reductase and saccharopine dehydrogenase activities in human kidney. 3 A role in lysine catabolism could justify the presence of the relatively high level of kynurenine aminotransferase activity in kidney 4,5 which lacks a complete pathway for tryptophan catabolism, a-Aminoadipate (kynurenine) aminotransferase also catalyzes aminotransfer of 3,5-diiodotyrosine (halogenated tyrosine aminotransferase) suggesting a possible role in thyroid hormone metabolism. 6 The reactions catalyzed by the kynurenine and halogenated tyrosine aminotransferase activities are illustrated in Fig. 1. ~-Aminoadipate (kynurenine):o~-ketoglutarate aminotransferase activity is also present in rat kidney mitochondria 6 and the supernatant 7 and 2 B. D. Manning and M. Mason, Life Sci. 17, 225 (1975). 3 j. Hutzler and J. Dancis, Biochim. Biophys. Acta 377, 42 (1975). 4 M. M a s o n , J. Biol. Chem. 211, 839 (1954). 5 M. Mason and C. P. Berg, J. Biol. Chem. 188, 783 (1951). 6 M. C. Tobes and M. Mason, Life Sci. 22, 793 (1978). 7 M. Tobes and M. Mason, Biochern. Biophys. Res. Commun. 62, 390 (1975).
666
O~-AMINOADIPATE
[83]
mitochondrial8 fractions of rat liver. The kidney6 and livers mitochondrial enzymes have been partially purified. L-a-Aminoadipate Aminotransferase Assay a
L - o< - A m i n o a d i p a t e
+ o<-Ketoglutarate
~
~ o<-Ketoadipate + G l u t a m a t e b
Activity of this enzyme can be measured from direction a or b by the rate of disappearance (direction a) or appearance (direction b) of a-ketoglutarate with glutamate dehydrogenase. 8 Reagents
Potassium phosphate, pH 7.5 Pyridoxal-5'-phosphate a-Ketoglutarate, pH 7.5 Potassium L-a-aminoadipate, 0.1 M, pH 7.5 HC1, 1 M KOH, 1 M Ammonium chloride NADH Glutamate dehydrogenase Procedure in the " a " Direction. The assay mixture contains 167 mM potassium phosphate buffer (pH 7.5), 0.26 mM pyridoxal-5'-phosphate, 1.67 mM a-ketoglutarate, and enzyme in a volume of 0.3 ml. After a 5 min incubation at 37°, the reaction is initiated by addition of 0.2 ml of 0.1 M potassium L-a-aminoadipate and incubated for 10 min at 37°. Incubation is terminated by addition of 0.1 ml 1 M HCI and the reaction mixture neutralized with 0.1 ml 1 M KOH. A 0.3-ml aliquot is analyzed for a-ketoglutarate. A unit of enzyme activity is defined as the amount of enzyme producing the disappearance of 1/zmol of a-ketoglutarate per min. The aketoglutarate that disappears is calculated by subtracting the a-ketoglutarate remaining from the a-ketoglutarate in a control containing no enzyme. Procedure in the " b " Direction. Assay conditions are the same as for the " a " direction except that a-ketoglutarate and L-a-aminoadipate were replaced by a-ketoadipate and L-glutamate. With this assay the reaction is linear with respect to time and enzyme concentration until 0.1/xmol of aketoglutarate is produced. A unit of enzyme activity is defined as the amount of enzyme producing 1 /xmol of a-ketoglutarate per min. 8 y. Nakatani, M. Fujioka, and K. Higashino,Biochim. Biophys. Acta 198, 219 (1970).
[83]
KYNURENINEAMINOTRANSFERASE
667
Tobes and Mason 7 report the use of this assay with some modifications which include (1) potassium phosphate buffer at pH 7.0 rather than 7.5, (2) neutralization after addition of 0.1 ml 1 M HC1 with 0.1 ml 1.1 M KOH to pH 7.5 rather than I M KOH to pH 7.0, and (3) centrifugation after neutralization at 5000 g for 5 min to remove precipitates. They also found that transferase activity in a mitochondrial suspension is increased by addition of CaC12 to 2 mM. The CaCI2 has no effect on glutamate dehydrogenase activity. Addition of CaCI2 does not increase transferase activity in the supernatant fraction of disrupted mitochondria. ct-Ketoglutarate Determination. a-Ketoglutarate is determined by the amount of N A D H oxidized in the presence of NH4 + and glutamate dehydrogenase according to the following equation: NADH * H + o<-Ketoglutarate 4- NH+4
~
NAD+ -~
> Glutamate
An incubation mixture contains 100 mM potassium phosphate buffer (pH 7.5), 50 mM NH4C1, 0.1 mM NADH, and 0.3 ml of neutralized aminotransferase reaction mixture in a final volume of 3.0 ml. The reaction is initiated by addition of glutamate dehydrogenase and the oxidation of N A D H followed spectrophotometrically at 340 nm. The molar absorptivity of N A D H at 340 nm is 6220. A unit of enzyme activity is defined as the amount of enzyme oxidizing 1 ~mol of NADH per min. Kynurenine Aminotransferase Assay L-Kynurenine + o< -Ketoglutarate
) Kynurenate + G l u t a m a t e
The activity of kynurenine aminotransferase is measured spectrophotometrically by following the appearance of kynurenate. 7
Reagents Pyridoxal-5'-phosphate a-Ketoglutarate, pH 6.5 Imidazole-HCi, pH 6.5 e-Kynurenine sulfate, pH 6.5 1% Boric acid in 95% ethanol Procedure. The incubation mixture contains 0.20 mM pyridoxal-5'phosphate, 5.00 mM a-ketoglutarate, 3.26 mM L-kynurenine sulfate, 100 mM imidazole-HCi buffer (pH 6.5), and 0 to 0.1 ml of enzyme solution in a final volume of 0.60 ml. The mixture is incubated at room temperature for 10 min before the reaction is initiated by addition of L-kynurenine sulfate. After a 30 min incubation at 37° the reaction is terminated by addition of
668
a-AMINOADIPATE
[83]
10 ml of ethanolic boric acid solution. Precipitates are removed by centrifugation at 5000 g for 5 min. Kynurenate formed is determined spectrophotometrically at 333 nm with correction for any kynurenine present by its absorption at 365 nm. Kynurenate absorbance is calculated according to the equation9: Absorbance at 333 nm due to kynurenate = (absorbance at 333 nm) - (0.39 x absorbance at 365 nm) The molar absorptivity of kynurenate at 333 nm is 8850.~° A unit of enzyme activity is defined as the amount of enzyme producing 1 /~mol of kynurenate per min. 3,5-Diiodotyrosine Aminotransferase Assay L - 3, 5 - D i i o d o t y r o s i n e + o< - K e t o g l u t a r a t e
> L-3,5
-Diiodo-4-hydroxyphenylpyruvate
+ Glutamat~
This transferase activity is determined by following the formation of the 3,5-diiodo-4-hydroxyphenylpyruvate product measured spectrophotometrically as the enol-borate complex.~l
Reagents Pyridoxal-5'-phosphate L-3,5-Diiodotyrosine, pH 7.0 or 6.5 Sodium phosphate, pH 7.0 or 6.5 a-Ketoglutarate, pH 7.0 or 6.5 H2SO4, 6 N Arsenate, 2 M, pH 6.5 Arsenate, 2 M which is I M in borate, pH 6.5
Procedure. The reaction mixture contains the 25 gM pyridoxal-5'phosphate, 2.5 mM L-3,5-diiodotyrosine, and 31.3 mM sodium phosphate buffer which is 0.31 mM in EDTA at pH 7.0 jl or 6.5, 6 and the enzyme in a final volume of 1.6 ml. After a 5 min incubation period at 37° the reaction is initiated by addition of a-ketoglutarate to 16 mM. The reaction is stopped, after incubation for 10 to 210 min at 37°, by addition of 0.1 ml of 6 N H2SO4. A 0.7-ml aliquot of the acidified incubation mixture is added to 2 ml of the arsenate solution (control). A second 0.7-ml aliquot is added to 2 ml of the arsenate-borate solution (enol-borate sample). Absorbance of the enol-borate sample is read at 330 nm. The molar absorptivity of the enol-borate complex of 3,5-diiodo-4-hydroxyphenylpyruvate at 330 nm is 9 M. Tobes, personal c o m m u n i c a t i o n . z0 W. E. K n o x , Biochem. J. 53, 379 (1953). H M. N a k a n o , J. Biol. Chem. 242, 73 (1967).
[83]
KYNURENINE AMINOTRANSFERASE
669
8110. H A unit of activity is defined as the amount of enzyme required for the formation of 1/zmol of 3,5-diiodo-4-hydroxyphenylpyruvateper min. Purification ~ All procedures are carried out at 0 to 4°. Solutions are prepared with redistilled and deionized water. Buffers are all potassium phosphate containing 10 mM 2-mercaptoethanol. Male adult albino rats (250 to 350 g) are sacrificed by decapitation and the kidneys immediately placed on ice. The kidneys are weighed, placed in a cold homogenizer in 4 ml of 0.25 M sucrose per g of tissue, homogenized, and centrifuged at 30,000 g for 20 min. 12,13 The supernatant is decanted and frozen immediately. The frozen supernatant is usually used within 3 weeks but loses little activity over a 3-month period at - 1 5 °. Kidneys from 30 rats yield approximately 200 ml of supernatant. Copurification of o~-aminoadipate and kynurenine aminotransferase activities is summarized in the table. Step I. A c i d Precipitation. Approximately 200 ml of frozen supernatant is thawed. The solution is made 10/zM in pyridoxal-5'-phosphate, titrated to pH 5.25 with 1.0 M acetic acid, and allowed to stand 5 min with occasional stirring. After centrifugation at 30,000 g for 10 min the supernatant is titrated to pH 6.5 with 1.0 M NaOH. Step 2. A m m o n i u m Sulfate and Heat Fractionation. Supernatant from the acid precipitation step is made 35% saturated with solid ammonium sulfate 14 and, while maintaining the pH at 6.5 with addition of 1.0 M NaOH, the suspension is stirred for 1 hr. The solution is made 0.05 mM in pyridoxal-5'-phosphate, taken to 55° for 5 min with constant stirring, brought back to 4° in an ice bath, and centrifuged at 30,000 g for 20 min. The supernatant is taken to 65% saturation in ammonium sulfate, stirred for 1 to 2 hr while maintaining the pH at 6.5 with 1.0 M NaOH, and the resulting suspension centrifuged at 30,000 g for 20 min. The supernatant is discarded and the precipitate dissolved in a minimal volume (20 to 24 ml) of 8 mM buffer at pH 7.1 and dialyzed against 2.1 liters of buffer for 19 hr with one change of buffer. Step 3. DEAE-Cellulose Chromatography. A 3.4 × 9.0 cm column is equilibrated with 8 mM buffer, pH 7.1, and the dialyzed solution applied to the column at approximately 45 ml/hr. Collecting 5 ml fractions, the enzyme is eluted off with the same buffer at the same flow rate. Aminotransferase activities which appear between fractions 40 to 60 are combined. 12M. Mason, J. Ford, and H. L. C. Wu, Ann. N.Y. Acad. Sci. 166(1), 170 (1969). ~3M. Mason, Biochem. Biophys. Res. Commun. 60, 64 (1974). 14M. Dixonand E. C. Webb, "Enzymes," 2nd ed., p. 40. AcademicPress, New York, 1964.
o
©
z
z
z
<~ z~ z
z
<
,,,
z
m
z
o
~z <
© z <
© Z
_o {.-
r, o
k~
[83]
KYNURENINE AMINOTRANSFERASE
671
Step 4. Hydroxylapatite I Chromatography. A 2.2 x 18.0 cm column is equilibrated with 8 mM buffer at pH 7.0 and the pooled DEAE eluate applied at 25 ml/hr. The column is eluted with 1.5 column volumes of 8 mM buffer and then eluted, at the same flow rate, with a linear gradient of 200 ml of 8 mM buffer, pH 7.0, and 200 ml of 210 mM buffer, pH 7.0. Fractions of 4 ml are collected and the active ones, from 117 to 128, are pooled and concentrated to 10 ml by means of a 50 ml Diaflo cell with a PM-10 membrane at an N2 pressure of 10 to 15 psi. The concentrated enzyme is dialyzed overnight with one change of buffer against 1 liter of 70 mM buffer at pH 7.0. Step 5. Hydroxylapatite H Chromatography. A 1.0 x 30.0 cm column is equilibrated with 70 mM buffer, pH 7.0, and the dialyzed sample applied at the rate of 7 ml/hr. Elution of the column is initiated with 1.5 column volumes of 70 mM buffer, pH 7.0, followed by a linear gradient elution of 94 ml of 70 mM buffer, pH 7.0, and 94 ml of 140 mM buffer at pH 7.0 at 8-9 ml/hr. Fractions of 2 ml are collected. The enzyme activity elutes off from fractions 65 to 75. With one change of buffer, the pooled active fractions are dialyzed overnight against 1 liter of 70 mM buffer, pH 7.0, containing 0.20 mM pyridoxal-5'-phosphate. Step 6. Hydroxylapatite III Chromatography. A 1.0 x 33.0 cm column is equilibrated with 70 mM buffer, pH 7.0, and the dialyzed sample applied at 5 ml/hr. The initial elution is with 1.5 column volumes of 70 mM buffer at pH 7.0 followed by elution of enzyme activity with a linear gradient of 100 ml of 70 mM buffer, pH 7.0, and 100 ml of 140 mM buffer, pH 7.0, at a rate of 4 to 5 ml/hr. Collecting 2 ml fractions, the enzyme activity elutes from fractions 75 to 85. The pooled active fractions are made 0.20 mM in pyridoxal-5'-phosphate and concentrated to 2 ml by ultrafiltration in a 10 ml Diaflo cell with a PM-10 membrane at a N2 pressure of 10 psi. After concentration, the enzyme is stored at 4° in 70 mM buffer, pH 7.0, that is 0.20 mM pyridoxal-5'-phosphate and 10 mM in 2-mercaptoethanol. The protein concentration of the stored enzyme is approximately !.0 mg/ml. After all chromatography steps the a-aminoadipate and kynurenine aminotransferase activities are dependent on added pyridoxal-5'-phosphate. Therefore, the enzyme purified as the apoenzyme. The overall purification with respect to the crude supernatant is 283fold for o~-aminoadipate aminotransferase activity and 294-fold for kynurenine aminotransferase activity. Purification relative to the crude homogenate is 600-fold. The enzyme is nearly homogeneous according to analytical disc gel electrophoresis at pH 8.9 and 7.5, isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
672
O~-AMINOADIPATE
[83]
Stability. The apoenzyme is most stable under the purification conditions in the pH range of 6.5 to 7.5. Maximal activity is dependent on the presence of a reducing agent such as 2-mercaptoethanol. The apoenzyme is quite stable in phosphate buffer, but when dialyzed against Tris-HC1 or imidazole-HC1, the enzyme activities are rapidly and irreversibly lost. In the Tris or imidazole buffers the apoenzyme is stable only in the presence of saturating levels of pyridoxal-5'-phosphate. Physical Characteristics The average molecular weight of the enzyme is 85,000. The average sedimentation coefficient is 5.8. No differences are observed for the sedimentation coefficient for sedimentation of apo- versus holoenzyme and crude versus purified enzyme. The enzyme is composed of two similar sized subunits (45,000 -+ 850) that are not linked by intrachain disulfide bonds. The isoelectric point of the enzyme is 6.56 -+ 0.06. Specificity
The Purified Enzyme from Rat Kidney. a-Aminoadipate is more reactive with the enzyme than kynurenine (see the table). While no data are presented, 1 the specificity with other amino acids, using a-ketoglutarate as the amino-acceptor, is described to be similar to those observed with a partially purified preparation from rat liver mitochondria. 8 The exception to this similarity is activity with aspartate (see the table). While the available data are consistent with activity with aspartate reflecting a slight contamination by aspartate aminotransferase, the possibility remains that this activity is intrinsic. Additional specificities reported are strong activity with 3,5-diiodotyrosine 6 and weak activities with tryptophan, phenylalanine, and tyrosine.15 The Partially Purified Enzyme from Rat Liver. The specificity of a partially purified preparation of a-aminoadipate : a-ketoglutarate aminotransferase from rat liver mitochondria has been reported. 8 On a relative activity scale in which L-a-aminoadipate is assigned an arbitrary value of 100 and in which a-ketoglutarate is used as the amino-acceptor, OL-aaminopimelate showed 14 and L-norleucine 15. Amino acids that are less than 2 include L-aspartate, L-alanine, L-a-aminobutyrate, glycine, Lvaline, L-leucine, L-isoleucine, L-norvaline, L-e-aminocaproate, Lthreonine, L-serine, L-homoserine, L-cysteine, L-methionine, L-lysine, Larginine, L-citrilline, L-ornithine, L-histidine, L-tyrosine, L-tryptophan, L-phenylalanine, L-proline, and L-glutamate. 15 M. C. Tobes, Ph.D. Thesis, University of Michigan, Ann Arbor (1976).