Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase

Andrzej Lewandowicz, Wuxian Shi, Gary B. Evans, Peter C. Tyler, Richard H. Furneaux, Luiz A. Basso, Diogenes S. Santos, Steven C. Almo, Vern L. Schramm

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 Å) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

Original languageEnglish (US)
Pages (from-to)6057-6066
Number of pages10
JournalBiochemistry
Volume42
Issue number20
DOIs
StatePublished - May 27 2003

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Purine-Nucleoside Phosphorylase
Mycobacterium tuberculosis
Crystal structure
Ions
Anions
Phosphates
Nucleophiles
Substrates
Cations
Carbon
DADMe-immucillin H
Enzymes
purine
forodesine

ASJC Scopus subject areas

  • Biochemistry

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Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase. / Lewandowicz, Andrzej; Shi, Wuxian; Evans, Gary B.; Tyler, Peter C.; Furneaux, Richard H.; Basso, Luiz A.; Santos, Diogenes S.; Almo, Steven C.; Schramm, Vern L.

In: Biochemistry, Vol. 42, No. 20, 27.05.2003, p. 6057-6066.

Research output: Contribution to journalArticle

Lewandowicz, Andrzej ; Shi, Wuxian ; Evans, Gary B. ; Tyler, Peter C. ; Furneaux, Richard H. ; Basso, Luiz A. ; Santos, Diogenes S. ; Almo, Steven C. ; Schramm, Vern L. / Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase. In: Biochemistry. 2003 ; Vol. 42, No. 20. pp. 6057-6066.
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AU - Tyler, Peter C.

AU - Furneaux, Richard H.

AU - Basso, Luiz A.

AU - Santos, Diogenes S.

AU - Almo, Steven C.

AU - Schramm, Vern L.

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N2 - Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 Å) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

AB - Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 Å) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

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