BACKGROUND
the aminotransferase capable of catalyzing the transamination of kynurenine to kynurenic acid  using various co-substrates, has commonly been termed kynurenine aminotransferase .
kyna is the only known endogenous antagonist of the n-methyl-d-aspartate subtype of glutamate receptors <cit> .
it is also an antagonist of the α7-nicotinic acetylcholine receptor <cit> .
in addition, kyna is identified as an endogenous ligand for an orphan g-protein-coupled receptor  that is predominantly expressed in immune cells <cit> .
abnormal concentration of kyna in cerebrospinal fluid/brain tissue has been observed in patients with mental and neurological disorders, including the huntington's disease, alzheimer's disease, schizophrenia, multiple sclerosis and others .
these data suggest that kyna, acting as an endogenous modulator of glutamatergic and cholinergic neurotransmission, may be functionally significant in the development and progression of these diseases.
in addition to its roles as an excitatory amino acid and α7-nicotinic acetylcholine antagonist, kyna is also involved in the control of the cardiovascular function by acting at the rostral ventrolateral medulla of the central nervous system  <cit> .
spontaneously hypertensive rat, the most widely used animal model for studying genetic hypertension, is associated with abnormally low kyna levels in the area of cns which controls physiological blood pressure <cit> .
kyna is produced enzymatically by irreversible transamination of kynurenine, the key intermediate in the tryptophan catabolic pathway.
in humans, rats and mice, four proteins arbitrarily named kat i, ii, iii and iv, have been considered to be involved in kyna synthesis in the cns <cit> .
kat i is identical to glutamine transaminase k  and cysteine conjugate beta-lyase  1; kat ii is identical to aminoadipate aminotransferase ; kat iii is identical to ccbl 2; and kat iv is identical to glutamic-oxaloacetic transaminase   <dig> and mitochondrial aspartate aminotransferase .
although the involvement of these enzymes in brain kyna production has been discussed, their specific roles in brain kyna synthesis remain to be established.
among the individual mammalian kats, kat i and kat iii share similar genomic structure and high sequence identity  <cit>  and therefore likely have overlapped biological functions.
an increase in kat i and kat iii expression was observed in kat- <dig> -/- mice brain, suggesting that kat i and kat iii expression compensated for the loss of kat ii  <cit> .
this also might explain why phenotypes such as the hyperactivity and abnormal motor coordination in the kat- <dig> -/- mice were rescued <cit> .
these data suggest the importance of mammalian kat i and kat iii in maintaining kyna level in kat- <dig> -/- mouse brain.
there have been many studies dealing with the biochemical characteristics of mammalian kat i and kat ii <cit> .
the crystal structures of human kat i  <cit>  and its homologues, glutamine-phenylpyruvate aminotransferase from thermus thermophilus hb <dig>  <cit>  and kat from a mosquito, aedes aegypti  <cit> , have been determined.
the crystal structure of human kat ii  <cit>  and its homologues from pyrococcus horikoshii  <cit>  and thermus thermophilus  <cit>  have also been determined.
the biochemical function and structural characteristics of mouse kat  iii have been determined <cit> ; and there have been a number of studies concerning the biochemical characterization of kat iv <cit> .
in this study, we functionally expressed mkat i, ii, iii, and iv in the same expression system, purified their recombinant proteins, investigated their ph optima, temperature preferences, and identified specific inhibitors for each individual mkat.
these properties of mkats i, ii, iii, and iv will help develop specific assays for the four enzymes, which could be used to identify which kat is affected in mouse disease models."
RESULTS
biophysical properties of mkats
sds-page analysis showed single band for each recombinant protein .
freezing and thawing did not alter the activity of the isolated enzymes.
mkat i showed maximum activity at  <dig> to 70°c , mkat ii showed maximum activity at 50°c , both mkat iii and mkat iv  showed maximum activity at 60°c.
the fact that all four enzymes showed high activity at 50°c, with mkat i and iv functioning at 70°c, indicates they are resistant to heating.
tm  values of mkat i, ii, iii, and iv are  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig> °c, respectively.
fig.
<dig> shows the fitting curve of each enzyme during the denaturation by heat treatment.
the calculated half life in first order kinetics  values of mkat i, ii, iii, and iv at 65°c are  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  min, respectively .
mkat i, ii, and iv all had a broad optimum ph range, while mkat iii had a relatively narrow optimum ph range.
ph profiles of the four kat proteins are all bell-shaped curves .
mkat i and iii showed maximum activity at ph  <dig> , mkat ii showed maximum activity at ph  <dig> to  <dig> , and mkat iv  showed maximum activity at ph  <dig>  the calculated pka values of mkat i, mkat ii, mkat iii, and mkat iv are  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
the calculated pkb values of mkat i, mkat ii, mkat iii, and mkat iv are  <dig>  ±  <dig> ,  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
comparison of the ph optima of mkat ii and human kat ii, indicates that the ph optimum of mkat ii is apparently shifted toward more acidic conditions, as it showed higher activity at ph  <dig> than at ph  <dig>  in contrast to human kat ii which showed much higher activity at ph  <dig> than at ph  <dig>  the activity of mkat ii at ph  <dig> is not shared by the other three mkats, which suggests a possible test for the specific activity of mkat ii.
the ph optimum of mkat i is similar to that of human kat i  <cit> , while the optimum of mkat iii expressed in escherichia coli is the same as that of mkat iii expressed in insect cells <cit> .
substrate specificities of mkat iv
mkat iv was tested for aminotransferase activity towards  <dig> different amino acids using α-ketoglutarate , as a primary amino group acceptor.
the enzyme showed the highest activity with aspartate and glutamate as substrates, but also showed activity with other amino acids, including some aromatic amino acids  and sulfur containing amino acids  .
effects of other amino acids on four mkats
the inhibition of each of  <dig> other amino acids was investigated by incorporating them individually into reaction mixtures containing  <dig> mm kynurenine and each of the mkats in turn.
of these, tryptophan, phenylalanine, glutamine, and cysteine were observed to inhibit mkat i activity; methionine, glutamine, histidine, cysteine, leucine, and phenylalanine to inhibit mkat iii activity; and, aspartate and glutamate to inhibit around 80% and tyrosine, phenylalanine, tryptophan, glutamine, cysteine, asparagine, and histidine to inhibit  <dig> - 40% of mkat iv activity.
although aminoadipate, lysine, 3-hk show inhibition of mkat ii activity, none of them inhibit more than 30% activity .
kat activities of mouse brain crude proteins
the brain crude proteins showed kat activity with all  <dig> tested α-ketoacids .
in particular, the crude proteins showed high activity with glyoxylate, a good co-substrate for mkat i and iii; and with α-ketoglutarate, a good substrate for mkat ii and iv.
these results are consistent with previous studies which demonstrated that mouse brains have all four kat enzymes <cit> .
based on the aforementioned results of amino acid inhibition study, we used methionine, tryptophan, aminoadipate, and aspartate to test inhibition of kat activity in mouse brain crude protein.
we found that aspartate inhibited 48% kat activity in the crude protein at ph  <dig>  using α-ketoglutarate as a co-substrate  and 30% activity at ph  <dig>  using glyoxylate as a co-substrate.
tryptophan inhibited 26% and methionine inhibited 15% kat activity at ph  <dig> using glyoxylate as a co-substrate .
aminoadipate did not significantly inhibit kat activity at either ph  <dig> or ph  <dig> .
discussion
the identity of asat with kat was first reported in e. coli  <cit> , and when later it was found that mitochondrial asats in mice, rats, and humans had kat activity, it was also named kat iv <cit> .
in this study, we showed mkat iv has high transamination activity towards glutamate and aspartate, and detectable activity towards phenylalanine, tyrosine, cysteine, tryptophan, 3-hk, methionine, kynurenine, and asparagine.
kat iv was considered as a major player for the formation of kyna in mouse, rat and human brains <cit> .
our results certainly show that mouse brain protein extract has high mkat iv activity as evidenced by the strong aspartate inhibition of the kyna formation in the reaction mixture.
high kat activity is detected in mouse brain crude proteins using α-ketoglutarate as a co-substrate at neutral ph condition, and as mkat iv is majorly responsible for this kat activity, its contribution in brain kyna synthesis has yet to be excluded.
a gene knock-out study in animal models could address this question definitively.
kyna is a metabolite in the kynurenine pathway, a major pathway in tryptophan metabolism, and its formation from tryptophan is a complicated process involving a number of enzymes  <cit> .
the overall process leading to the formation of kyna includes oxidation of tryptophan to formylkynurenine catalyzed by tryptophan  <dig>  dioxygenase and indoleamine  <dig>  dioxygenase; hydrolysis of formylkynurenine to kynurenine catalyzed by kynurenine formamidase; and transamination of kynurenine to kyna catalyzed by four kats.
alternatively, kynurenine could be oxidized by kynurenine 3-monooxygenase to form 3-hk or hydrolyzed by kynureninase to form anthranilic acid.
the mouse brain crude extract not only consists of four kat enzymes but also other proteins involved in the kynurenine pathway.
in our preliminary studies, when we tested kat activity at ph  <dig> , we observed the formation of 3-hk and anthranilic acid ; we also noticed that kyna formation was increased by adding tryptophan to the reaction mixture.
this is perhaps because indoleamine  <dig>  dioxygenase and tryptophan  <dig>  dioxygenase oxidize tryptophan at the start of the kynurenine pathway, increasing the kynurenine concentration, which, in turn, results in increased kyna formation.
since kynurenine 3-monooxygenase oxidizes kynurenine to 3-hk and kynureninase hydrolyzes kynurenine to anthranilic acid, they may compete with the mkats for the same substrate, kynurenine, thus interfering with the kat activity assay.
the brain crude extract sample also contains amino acids and α-keto acids, which are substrates or inhibitors of four mkats; nadh and nadph, which are cofactors of kynurenine 3-monooxygenase; and hemin, which functions as a cofacor of both indoleamine  <dig>  dioxygenase and tryptophan  <dig>  dioxygenase.
the presence of these substances and other kynurenine pathway enzymes could directly affect kat activity assay by providing a co-substrate for kat enzymes or by competitively inhibiting the enzymes; or they could indirectly change kat activity by enhancing the activity of kynureninase or kynurenine 3-monooxygenase which competitively use kynurenine.
to prevent or decrease the interference from other kynurenine pathway enzymes and brain chemicals, we first dialyzed the brain crude sample with a  <dig> kda molecular weight cutoff membrane to eliminate possible substrates and inhibitors of four kat enzymes, and cofactors of other kynurenine pathway enzymes from the sample.
herein, we demonstrated all mkat i, iii, iv showed high resistance to heat treatment, and mkat ii also showed noticeable activity at 60°c.
the tm value of mkat ii is similar to that of other mkats except that its activity declines at 60°c, which is lower than the tm value.
one possible reason may be that the cofactor binding or dimer interaction is disrupted at 60°c, without changing the absorbance at  <dig> nm significantly.
spectral changes in  <dig> nm caused by protein denaturation are attributed to changes in the environment of tyrosyl and tryptophyl side chains, which are buried in the hydrophobic region of the native protein molecule  <cit> .
so the contribution of this spectral change is made mainly by the disruption of secondary and tertiary structures.
the damage of quaternary structure may not significantly contribute to this change unless there are enough tyrosine and tryptophan residues involved in dimerization.
comparing the crystal structures of the four mkats will help elucidate the mechanism.
cd spectroscopy of mouse indoleamine  <dig>  dioxygenase indicated that the tm value is 60°c, with a second intermediate tm at 44°c <cit> , and that rabbit indoleamine  <dig>  dioxygenase was not stable at 55°c <cit> .
mosquito kynurenine 3-monooxygenase shows very little activity at 55°c <cit> , but there is little information about the thermal stability of mammalian kynurenine 3-monooxygenases.
here, we did notice that 3-hk formation is almost undetectable in the reaction mixture at ph  <dig>  after dialysis and heat treatment of brain crude extract at 60°c for  <dig> min .
apparently, kynurenine 3-monooxygenase was deactivated either by taking away a cofactor or by destroying the secondary, tertiary or quaternary structure with heat treatment.
mammalian kynureninase has been studied for its substrate specificity, ph optimum and inhibition <cit> , but there is little information about its thermal stability.
the heat treatment of the crude extract at 60°c for  <dig> min did decrease the anthranilic acid production catalyzed by kynureninase .
however, further study of the biochemical and biophysical properties of mouse kynureninase, indoleamine  <dig>  dioxygenase, tryptophan  <dig>  dioxygenase, and kynurenine 3-monooxygenase is needed in order to avoid the interferences with the kat specific assays from these kynurenine pathway enzymes.
the purpose of comparing four different mkats is to find their unique characteristics and to develop a sensitive and specific method to assay each mkat.
the assay previously reported for kat i using pyruvate as a co-substrate at ph  <dig>   <cit>  would also detect activity of the other three kats, because they all can use pyruvate as an amino group acceptor and have activity at this ph <cit> .
in addition, pyruvate is not a favored co-substrate of kat i. the commonly used kat ii assay  <cit>  is also problematic because under the same assay conditions, kat i and iii have detectable activity and kat iv has high activity <cit> .
mkat enzymes might not have exactly the same properties as their human homologues, which have been characterized previously  <cit> , we used the same protein expression system to produce all four mkats and obtained ph profiles, temperature preferences and enzyme inhibitions for each individual enzyme.
some of the results are interesting, e.g.
optimum ph of mkat ii  is different from that of human kat ii   <cit> .
the characteristics of each mkat, which could be used for developing specific assays of four mkat enzymes are summarized for clarity in table  <dig>  based on this information, we did preliminary tests to develop a specific assay of each kat.
the assays for mkat i and mkat ii were shown to be highly specific; and the assay methods for mkat iii and iv were shown to be specific .
specific
tm: the midpoint temperature of the protein melting; t1/2: half life value at 65°c
CONCLUSIONS
this is the first kinetic characterization of the temperature and ph effects of the four mkat enzymes.
the characteristics reported here could be used to develop specific assay methods for each of the four murine kats.
these specific assays could be used to identify which kat is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of kat-involved human diseases.

 the aminotransferase capable of catalyzing the transamination of kynurenine to kynurenic acid using various co-substrates, has commonly been termed kynurenine aminotransferase. kyna is the only known endogenous antagonist of the n-methyl-d-aspartate subtype of glutamate receptors. it is also an antagonist of the α7-nicotinic acetylcholine receptor. in addition, kyna is identified as an endogenous ligand for an orphan g-protein-coupled receptor that is predominantly expressed in immune cells. abnormal concentration of kyna in cerebrospinal fluid/brain tissue has been observed in patients with mental and neurological disorders, including the huntington's disease, alzheimer's disease, schizophrenia, multiple sclerosis and others. these data suggest that kyna, acting as an endogenous modulator of glutamatergic and cholinergic neurotransmission, may be functionally significant in the development and progression of these diseases. in addition to its roles as an excitatory amino acid and α7-nicotinic acetylcholine antagonist, kyna is also involved in the control of the cardiovascular function by acting at the rostral ventrolateral medulla of the central nervous system. spontaneously hypertensive rat, the most widely used animal model for studying genetic hypertension, is associated with abnormally low kyna levels in the area of cns which controls physiological blood pressure. kyna is produced enzymatically by irreversible transamination of kynurenine, the key intermediate in the tryptophan catabolic pathway. in humans, rats and mice, four proteins arbitrarily named kat i, ii, iii and iv, have been considered to be involved in kyna synthesis in the cns. kat i is identical to glutamine transaminase k and cysteine conjugate beta-lyase 1; kat ii is identical to aminoadipate aminotransferase; kat iii is identical to ccbl 2; and kat iv is identical to glutamic-oxaloacetic transaminase and mitochondrial aspartate aminotransferase. although the involvement of these enzymes in brain kyna production has been discussed, their specific roles in brain kyna synthesis remain to be established. among the individual mammalian kats, kat i and kat iii share similar genomic structure and high sequence identity and therefore likely have overlapped biological functions. an increase in kat i and kat iii expression was observed in kat- -/- mice brain, suggesting that kat i and kat iii expression compensated for the loss of kat ii. this also might explain why phenotypes such as the hyperactivity and abnormal motor coordination in the kat- -/- mice were rescued. these data suggest the importance of mammalian kat i and kat iii in maintaining kyna level in kat- -/- mouse brain. there have been many studies dealing with the biochemical characteristics of mammalian kat i and kat ii. the crystal structures of human kat i and its homologues, glutamine-phenylpyruvate aminotransferase from thermus thermophilus hb and kat from a mosquito, aedes aegypti, have been determined. the crystal structure of human kat ii and its homologues from pyrococcus horikoshii and thermus thermophilus have also been determined. the biochemical function and structural characteristics of mouse kat iii have been determined; and there have been a number of studies concerning the biochemical characterization of kat iv. in this study, we functionally expressed mkat i, ii, iii, and iv in the same expression system, purified their recombinant proteins, investigated their ph optima, temperature preferences, and identified specific inhibitors for each individual mkat. these properties of mkats i, ii, iii, and iv will help develop specific assays for the four enzymes, which could be used to identify which kat is affected in mouse disease models."  biophysical properties of mkats sds-page analysis showed single band for each recombinant protein. freezing and thawing did not alter the activity of the isolated enzymes. mkat i showed maximum activity at to 70°c, mkat ii showed maximum activity at 50°c, both mkat iii and mkat iv showed maximum activity at 60°c. the fact that all four enzymes showed high activity at 50°c, with mkat i and iv functioning at 70°c, indicates they are resistant to heating. tm values of mkat i, ii, iii, and iv are ±, ±, ±, ± °c, respectively. fig. shows the fitting curve of each enzyme during the denaturation by heat treatment. the calculated half life in first order kinetics values of mkat i, ii, iii, and iv at 65°c are ±, ±, ± and ± min, respectively. mkat i, ii, and iv all had a broad optimum ph range, while mkat iii had a relatively narrow optimum ph range. ph profiles of the four kat proteins are all bell-shaped curves. mkat i and iii showed maximum activity at ph, mkat ii showed maximum activity at ph to, and mkat iv showed maximum activity at ph the calculated pka values of mkat i, mkat ii, mkat iii, and mkat iv are ±, ±, ± and ±, respectively. the calculated pkb values of mkat i, mkat ii, mkat iii, and mkat iv are ±, ±, ± and ±, respectively. comparison of the ph optima of mkat ii and human kat ii, indicates that the ph optimum of mkat ii is apparently shifted toward more acidic conditions, as it showed higher activity at ph than at ph in contrast to human kat ii which showed much higher activity at ph than at ph the activity of mkat ii at ph is not shared by the other three mkats, which suggests a possible test for the specific activity of mkat ii. the ph optimum of mkat i is similar to that of human kat i, while the optimum of mkat iii expressed in escherichia coli is the same as that of mkat iii expressed in insect cells. substrate specificities of mkat iv mkat iv was tested for aminotransferase activity towards different amino acids using α-ketoglutarate, as a primary amino group acceptor. the enzyme showed the highest activity with aspartate and glutamate as substrates, but also showed activity with other amino acids, including some aromatic amino acids and sulfur containing amino acids. effects of other amino acids on four mkats the inhibition of each of other amino acids was investigated by incorporating them individually into reaction mixtures containing mm kynurenine and each of the mkats in turn. of these, tryptophan, phenylalanine, glutamine, and cysteine were observed to inhibit mkat i activity; methionine, glutamine, histidine, cysteine, leucine, and phenylalanine to inhibit mkat iii activity; and, aspartate and glutamate to inhibit around 80% and tyrosine, phenylalanine, tryptophan, glutamine, cysteine, asparagine, and histidine to inhibit - 40% of mkat iv activity. although aminoadipate, lysine, 3-hk show inhibition of mkat ii activity, none of them inhibit more than 30% activity. kat activities of mouse brain crude proteins the brain crude proteins showed kat activity with all tested α-ketoacids. in particular, the crude proteins showed high activity with glyoxylate, a good co-substrate for mkat i and iii; and with α-ketoglutarate, a good substrate for mkat ii and iv. these results are consistent with previous studies which demonstrated that mouse brains have all four kat enzymes. based on the aforementioned results of amino acid inhibition study, we used methionine, tryptophan, aminoadipate, and aspartate to test inhibition of kat activity in mouse brain crude protein. we found that aspartate inhibited 48% kat activity in the crude protein at ph using α-ketoglutarate as a co-substrate and 30% activity at ph using glyoxylate as a co-substrate. tryptophan inhibited 26% and methionine inhibited 15% kat activity at ph using glyoxylate as a co-substrate. aminoadipate did not significantly inhibit kat activity at either ph or ph. discussion the identity of asat with kat was first reported in e. coli, and when later it was found that mitochondrial asats in mice, rats, and humans had kat activity, it was also named kat iv. in this study, we showed mkat iv has high transamination activity towards glutamate and aspartate, and detectable activity towards phenylalanine, tyrosine, cysteine, tryptophan, 3-hk, methionine, kynurenine, and asparagine. kat iv was considered as a major player for the formation of kyna in mouse, rat and human brains. our results certainly show that mouse brain protein extract has high mkat iv activity as evidenced by the strong aspartate inhibition of the kyna formation in the reaction mixture. high kat activity is detected in mouse brain crude proteins using α-ketoglutarate as a co-substrate at neutral ph condition, and as mkat iv is majorly responsible for this kat activity, its contribution in brain kyna synthesis has yet to be excluded. a gene knock-out study in animal models could address this question definitively. kyna is a metabolite in the kynurenine pathway, a major pathway in tryptophan metabolism, and its formation from tryptophan is a complicated process involving a number of enzymes. the overall process leading to the formation of kyna includes oxidation of tryptophan to formylkynurenine catalyzed by tryptophan dioxygenase and indoleamine dioxygenase; hydrolysis of formylkynurenine to kynurenine catalyzed by kynurenine formamidase; and transamination of kynurenine to kyna catalyzed by four kats. alternatively, kynurenine could be oxidized by kynurenine 3-monooxygenase to form 3-hk or hydrolyzed by kynureninase to form anthranilic acid. the mouse brain crude extract not only consists of four kat enzymes but also other proteins involved in the kynurenine pathway. in our preliminary studies, when we tested kat activity at ph, we observed the formation of 3-hk and anthranilic acid; we also noticed that kyna formation was increased by adding tryptophan to the reaction mixture. this is perhaps because indoleamine dioxygenase and tryptophan dioxygenase oxidize tryptophan at the start of the kynurenine pathway, increasing the kynurenine concentration, which, in turn, results in increased kyna formation. since kynurenine 3-monooxygenase oxidizes kynurenine to 3-hk and kynureninase hydrolyzes kynurenine to anthranilic acid, they may compete with the mkats for the same substrate, kynurenine, thus interfering with the kat activity assay. the brain crude extract sample also contains amino acids and α-keto acids, which are substrates or inhibitors of four mkats; nadh and nadph, which are cofactors of kynurenine 3-monooxygenase; and hemin, which functions as a cofacor of both indoleamine dioxygenase and tryptophan dioxygenase. the presence of these substances and other kynurenine pathway enzymes could directly affect kat activity assay by providing a co-substrate for kat enzymes or by competitively inhibiting the enzymes; or they could indirectly change kat activity by enhancing the activity of kynureninase or kynurenine 3-monooxygenase which competitively use kynurenine. to prevent or decrease the interference from other kynurenine pathway enzymes and brain chemicals, we first dialyzed the brain crude sample with a kda molecular weight cutoff membrane to eliminate possible substrates and inhibitors of four kat enzymes, and cofactors of other kynurenine pathway enzymes from the sample. herein, we demonstrated all mkat i, iii, iv showed high resistance to heat treatment, and mkat ii also showed noticeable activity at 60°c. the tm value of mkat ii is similar to that of other mkats except that its activity declines at 60°c, which is lower than the tm value. one possible reason may be that the cofactor binding or dimer interaction is disrupted at 60°c, without changing the absorbance at nm significantly. spectral changes in nm caused by protein denaturation are attributed to changes in the environment of tyrosyl and tryptophyl side chains, which are buried in the hydrophobic region of the native protein molecule. so the contribution of this spectral change is made mainly by the disruption of secondary and tertiary structures. the damage of quaternary structure may not significantly contribute to this change unless there are enough tyrosine and tryptophan residues involved in dimerization. comparing the crystal structures of the four mkats will help elucidate the mechanism. cd spectroscopy of mouse indoleamine dioxygenase indicated that the tm value is 60°c, with a second intermediate tm at 44°c, and that rabbit indoleamine dioxygenase was not stable at 55°c. mosquito kynurenine 3-monooxygenase shows very little activity at 55°c, but there is little information about the thermal stability of mammalian kynurenine 3-monooxygenases. here, we did notice that 3-hk formation is almost undetectable in the reaction mixture at ph after dialysis and heat treatment of brain crude extract at 60°c for min. apparently, kynurenine 3-monooxygenase was deactivated either by taking away a cofactor or by destroying the secondary, tertiary or quaternary structure with heat treatment. mammalian kynureninase has been studied for its substrate specificity, ph optimum and inhibition, but there is little information about its thermal stability. the heat treatment of the crude extract at 60°c for min did decrease the anthranilic acid production catalyzed by kynureninase. however, further study of the biochemical and biophysical properties of mouse kynureninase, indoleamine dioxygenase, tryptophan dioxygenase, and kynurenine 3-monooxygenase is needed in order to avoid the interferences with the kat specific assays from these kynurenine pathway enzymes. the purpose of comparing four different mkats is to find their unique characteristics and to develop a sensitive and specific method to assay each mkat. the assay previously reported for kat i using pyruvate as a co-substrate at ph would also detect activity of the other three kats, because they all can use pyruvate as an amino group acceptor and have activity at this ph. in addition, pyruvate is not a favored co-substrate of kat i. the commonly used kat ii assay is also problematic because under the same assay conditions, kat i and iii have detectable activity and kat iv has high activity. mkat enzymes might not have exactly the same properties as their human homologues, which have been characterized previously, we used the same protein expression system to produce all four mkats and obtained ph profiles, temperature preferences and enzyme inhibitions for each individual enzyme. some of the results are interesting, e.g. optimum ph of mkat ii is different from that of human kat ii. the characteristics of each mkat, which could be used for developing specific assays of four mkat enzymes are summarized for clarity in table based on this information, we did preliminary tests to develop a specific assay of each kat. the assays for mkat i and mkat ii were shown to be highly specific; and the assay methods for mkat iii and iv were shown to be specific. specific tm: the midpoint temperature of the protein melting; t1/2: half life value at 65°c  this is the first kinetic characterization of the temperature and ph effects of the four mkat enzymes. the characteristics reported here could be used to develop specific assay methods for each of the four murine kats. these specific assays could be used to identify which kat is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of kat-involved human diseases. 
the characteristics reported here could be used to develop specific assay methods for each of the four murine kats. because of the substrate overlap of the four kat enzymes, it is difficult to assay the specific activity of each kat in animal brains. these specific assays could be used to identify which kat is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of kat-involved human diseases. midpoint temperatures of the protein melting, half life values at 65°c, and pka values of mouse kat i, ii, iii, and iv were,, and °c;,, and min; ph,, and, respectively. ph optima, temperature preferences of four kats were also tested in this study. kyna is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of n-methyl-d-aspartate receptors. four kat enzymes, kat i/glutamine transaminase k/cysteine conjugate beta-lyase kat ii/aminoadipate aminotransferase, kat iii/cysteine conjugate beta-lyase and kat iv/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. this study concerns the functional expression and comparative characterization of kat i, ii, iii, and iv from mice. at the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse kat i and iv, equimolar methionine inhibited only mouse kat iii and equimolar aspartate inhibited only mouse kat iv. the activity of mouse kat ii was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. kynurenine aminotransferase catalyzes the transamination of kynunrenine to kynurenic acid.
 kynurenine aminotransferase catalyzes the transamination of kynunrenine to kynurenic acid. kyna is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of n-methyl-d-aspartate receptors. four kat enzymes, kat i/glutamine transaminase k/cysteine conjugate beta-lyase kat ii/aminoadipate aminotransferase, kat iii/cysteine conjugate beta-lyase and kat iv/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. because of the substrate overlap of the four kat enzymes, it is difficult to assay the specific activity of each kat in animal brains.  this study concerns the functional expression and comparative characterization of kat i, ii, iii, and iv from mice. at the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse kat i and iv, equimolar methionine inhibited only mouse kat iii and equimolar aspartate inhibited only mouse kat iv. the activity of mouse kat ii was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. ph optima, temperature preferences of four kats were also tested in this study. midpoint temperatures of the protein melting, half life values at 65°c, and pka values of mouse kat i, ii, iii, and iv were,, and °c;,, and min; ph,, and, respectively.  the characteristics reported here could be used to develop specific assay methods for each of the four murine kats. these specific assays could be used to identify which kat is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of kat-involved human diseases. 