Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) Primary Lineweaver Burk (L/B) plot of kinetic data for inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (■); I = 50 nM (▲) I = 100 nM (▼); I = 200 nM (◀ ▶); I = 400 nM (●); I = 600 nM (□)) depicting mixed inhibition. s = substrate (3-hydroxykynurenine) and v = specific activity. The inset is a secondary plot of slope against [I] to determine the Ki (40 nM). The slopes were calculated from a L/B plot (n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC223355&req=5

Figure 4: Inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) Primary Lineweaver Burk (L/B) plot of kinetic data for inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (■); I = 50 nM (▲) I = 100 nM (▼); I = 200 nM (◀ ▶); I = 400 nM (●); I = 600 nM (□)) depicting mixed inhibition. s = substrate (3-hydroxykynurenine) and v = specific activity. The inset is a secondary plot of slope against [I] to determine the Ki (40 nM). The slopes were calculated from a L/B plot (n = 3).

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 rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) Primary Lineweaver Burk (L/B) plot of kinetic data for inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (■); I = 50 nM (▲) I = 100 nM (▼); I = 200 nM (◀ ▶); I = 400 nM (●); I = 600 nM (□)) depicting mixed inhibition. s = substrate (3-hydroxykynurenine) and v = specific activity. The inset is a secondary plot of slope against [I] to determine the Ki (40 nM). The slopes were calculated from a L/B plot (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC223355&req=5

Figure 4: Inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) Primary Lineweaver Burk (L/B) plot of kinetic data for inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (■); I = 50 nM (▲) I = 100 nM (▼); I = 200 nM (◀ ▶); I = 400 nM (●); I = 600 nM (□)) depicting mixed inhibition. s = substrate (3-hydroxykynurenine) and v = specific activity. The inset is a secondary plot of slope against [I] to determine the Ki (40 nM). The slopes were calculated from a L/B plot (n = 3).
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