Characterization of human brain kynurenine aminotransferases using [3H]kynurenine as a substrate

Schmidt, Werner; Guidetti, Paolo; Okuno, Etsuo; Schwarcz, Robert

doi:10.1016/0306-4522(93)90464-q

Cited by 44 publications

(46 citation statements)

References 20 publications

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“…When the phosphate and borate buffer mixture, adjusted to pH 6.0 to 11.0, was used to prepare mKAT III-kynurenine-glyoxylate reaction mixtures, mKAT III displayed optimum activity around pH 9.0 to 10.0 (Fig. 1B), which is close to the optimum pH reported in the literature (3,4,16,49) for mammalian brain KAT I but apparently different from the optimum pH (7.5 to 9.0) of recombinant hKAT I (25). mKAT III was tested for aminotransferase activity toward 24 different amino acids, using glyoxylate as a primary amino group acceptor.…”

Section: Resultssupporting

confidence: 81%

“…3), suggesting that 3-HK is a substrate of mKAT III. Based on these results, it seems apparent that the biochemical characteristics of mKAT III are similar to those described for mammalian brain KAT I (16,49).…”

Section: Discussionsupporting

confidence: 71%

“…The availability of highly purified recombinant hKAT I and mKAT III enabled us to critically compare the biochemical characteristics of these two enzymes. In a previous study, we determined that hKAT I was able to efficiently catalyze kynurenine to KNYA under physiological conditions, but it did not show any activity toward 3-HK (25) and differed from other reports (16,40,49) concerning mammalian KAT I. Comparison of the pH profiles of hKAT I and mKAT III clearly indicates that mKAT III is more active than hKAT I under basic conditions.…”

Section: Discussionmentioning

confidence: 81%

“…Optimum pHs of 9.5 to 10 for KAT I activity were seen in isolated human brain (3,4,16,49) and placenta (39), whereas optimal low pHs of 6.5 to 9 for "KAT I" activity were reported for insect cell-expressed hKAT I (pH 7.5 to 9.0) (25), rat liver (pH 6.5) (55), and heart (pH 8 to 9) (2). Since KAT I is a highly conserved protein, it is unlikely that the isolated KAT I enzyme, when tested in vitro, will have a different optimum pH.…”

Section: Discussionmentioning

confidence: 99%

“…These studies suggest the importance of mammalian KAT I and KAT III in maintaining the KYNA level in the kat-2 Ϫ/Ϫ mouse brain. Although many studies have dealt with the biochemical characteristics of mammalian KAT I and KAT II (3,4,9,16,19,25,49), little is known about the biochemical activity of KAT III. Likewise, the crystal structures of human KAT I (hKAT I) (48) and its homologues, glutamine-phenylpyruvate aminotransferase from Thermus thermophilus HB8 (13) and KAT from a mosquito, Aedes aegypti (AeKAT) (22), have been determined, as well as the crystal structure of hKAT II (26,47) and its homologue from Pyrococcus horikoshii (7).…”

mentioning

confidence: 99%

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Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Han

Robinson

Cai

et al. 2009

Molecular and Cellular Biology

111

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Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60°C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

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Section: Resultssupporting

confidence: 81%

Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Han

Robinson

Cai

et al. 2009

Molecular and Cellular Biology

111

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Characterization of rat brain kynurenine aminotransferases I and II

1997

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The endogenous neuroprotectant kynurenic acid (KYNA) is produced by irreversible transamination of L-kynurenine (KYN). In the brain, two distinct kynurenine aminotransferases (KAT I and KAT II) are responsible for the formation of KYNA. The present experiments were designed to examine the respective roles of the two KATs in the normal rat brain. To this end, the two enzymes were partially purified, and their characteristics were examined. KAT I (identical with glutamine transaminase K) had an optimal pH of 9.5, preferred pyruvate as a cosubstrate and was potently inhibited by glutamine. KAT II (identical with L-alpha-aminoadipate transaminase) had a neutral optimal pH, showed no preference for pyruvate, and was essentially insensitive to inhibition by glutamine. KAT II was selectively inhibited by quisqualic acid (IC50: 520 microM). The endogenous substrate 3-hydroxykynurenine had an approximately 10-fold preference for KAT II. The distinct properties of the two enzymes made it possible to measure brain KAT I and KAT II in parallel by using dialyzed tissue homogenate (to remove interfering endogenous amino acids). Under these conditions, both enzymes presented essentially the same apparent Km values as the partially purified enzymes. In lesioned, neurondepleted brain tissue and in brain regions other than the cerebellum, KYNA derived primarily from KAT II at physiologic pH. In summary, the present study describes a simple methodology for the simultaneous determination of the two KYNA-producing enzymes in small rat brain tissue samples and provides baseline values for future work in experimentally challenged animals.

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