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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.

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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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Reaction catalysed by kynureninase
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Figure 1: Reaction catalysed by kynureninase

Mentions: Kynureninase (EC 3.7.1.1) is a pyridoxal-5'-phosphate (PLP) dependent enzyme which catalyses the β, γ-hydrolytic cleavage of the amino acids kynurenine (1, R = H) and 3-hydroxykynurenine (1, R = OH) to give either anthranilic acid (2, R = H) or 3-hydroxyanthranilic acid (2, R=OH) and alanine (3) (Figure 1) [1].


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)

Reaction catalysed by kynureninase
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Reaction catalysed by kynureninase
Mentions: Kynureninase (EC 3.7.1.1) is a pyridoxal-5'-phosphate (PLP) dependent enzyme which catalyses the β, γ-hydrolytic cleavage of the amino acids kynurenine (1, R = H) and 3-hydroxykynurenine (1, R = OH) to give either anthranilic acid (2, R = H) or 3-hydroxyanthranilic acid (2, R=OH) and alanine (3) (Figure 1) [1].

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