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Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

Walsh HA, O'Shea KC, Botting NP - BMC Biochem. (2003)

Bottom Line: The potency of the various inhibitors was found to be species specific.The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase.The modified purification described is relatively quick, simple and cost effective.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemistry, University of St, Andrews, St Andrews, Fife, KY16 9ST UK. haw2@st-andrews.ac.uk

ABSTRACT

Background: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim.

Results: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed.

Conclusion: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective.

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Inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) Dixon plot illustrating inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (◆); I = 200 nM (▼); I = 400 nM (▲); I = 600 nM (■). The inhibition is mixed and gives a KI = 100 nM and the graph is a replot of the data (n = 3) used to construct a Michael's–Menten plot. The inset is a secondary plot of slope (from L/B) against inhibitor concentration for recombinant human kynureninase (n= 3 and r2 = 0.99) to calculate KI (5 nM). The concentration of substrate was varied between 2.5 μM – 20 μM. Lines were fitted to a straight line equation for linear regression.
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Figure 5: Inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) Dixon plot illustrating inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (◆); I = 200 nM (▼); I = 400 nM (▲); I = 600 nM (■). The inhibition is mixed and gives a KI = 100 nM and the graph is a replot of the data (n = 3) used to construct a Michael's–Menten plot. The inset is a secondary plot of slope (from L/B) against inhibitor concentration for recombinant human kynureninase (n= 3 and r2 = 0.99) to calculate KI (5 nM). The concentration of substrate was varied between 2.5 μM – 20 μM. Lines were fitted to a straight line equation for linear regression.

Mentions: From the results obtained in table 1 it is clear that there is significant difference in the degree of inhibition with the hydroxylated when compared to the methoxylated inhibitor. The type of inhibition is also mixed in all instances as indicated by the Lineweaver-Burk (Figure 4) and Dixon plots (Figure 5) [7]. Previously [8] it has been shown that the recombinant human enzyme is also inhibited similarly when treated with 3,5-dihydroxydesaminokynurenine, which was also the case with both inhibitors, used in this study.


Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

Walsh HA, O'Shea KC, Botting NP - BMC Biochem. (2003)

Inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) Dixon plot illustrating inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (◆); I = 200 nM (▼); I = 400 nM (▲); I = 600 nM (■). The inhibition is mixed and gives a KI = 100 nM and the graph is a replot of the data (n = 3) used to construct a Michael's–Menten plot. The inset is a secondary plot of slope (from L/B) against inhibitor concentration for recombinant human kynureninase (n= 3 and r2 = 0.99) to calculate KI (5 nM). The concentration of substrate was varied between 2.5 μM – 20 μM. Lines were fitted to a straight line equation for linear regression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC223355&req=5

Figure 5: Inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) Dixon plot illustrating inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (◆); I = 200 nM (▼); I = 400 nM (▲); I = 600 nM (■). The inhibition is mixed and gives a KI = 100 nM and the graph is a replot of the data (n = 3) used to construct a Michael's–Menten plot. The inset is a secondary plot of slope (from L/B) against inhibitor concentration for recombinant human kynureninase (n= 3 and r2 = 0.99) to calculate KI (5 nM). The concentration of substrate was varied between 2.5 μM – 20 μM. Lines were fitted to a straight line equation for linear regression.
Mentions: From the results obtained in table 1 it is clear that there is significant difference in the degree of inhibition with the hydroxylated when compared to the methoxylated inhibitor. The type of inhibition is also mixed in all instances as indicated by the Lineweaver-Burk (Figure 4) and Dixon plots (Figure 5) [7]. Previously [8] it has been shown that the recombinant human enzyme is also inhibited similarly when treated with 3,5-dihydroxydesaminokynurenine, which was also the case with both inhibitors, used in this study.

Bottom Line: The potency of the various inhibitors was found to be species specific.The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase.The modified purification described is relatively quick, simple and cost effective.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemistry, University of St, Andrews, St Andrews, Fife, KY16 9ST UK. haw2@st-andrews.ac.uk

ABSTRACT

Background: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim.

Results: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed.

Conclusion: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective.

Show MeSH
Related in: MedlinePlus