Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase

David W. Parkin, Vern L. Schramm

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The transition-state structure for inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitors with observed Km/Ki values up to 2 × 105 [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262]. Although slow-onset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The Km value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6-10.5. Dependencies of Vmax and Vmax/Km on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1-(S)-Phenyl-1,4-dideoxy-1,4-imino-D-ribitol (phenyliminoribitol) binds with an equilibrium Kd of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the Vmax profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding. It has been proposed that the positive charge of protonated inhibitor (pK 6.5) is a recognition feature for binding as a transition-state inhibitor. However, the pH analysis indicates that the neutral inhibitor is the preferred species for binding the active form of the enzyme. The slow-onset phase of phenyliminoribitol binding disappears at low pH, suggesting that a time-dependent protonation of the bound complex could be responsible for the slow-onset phase of inhibition.

Original languageEnglish (US)
Pages (from-to)13961-13966
Number of pages6
JournalBiochemistry
Volume34
Issue number42
StatePublished - 1995

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N-Glycosyl Hydrolases
Protonation
Inosine
Uridine
Substrates
Nucleophiles
Biochemistry
Hypoxanthine
Crithidia fasciculata
Catalysis
Catalyst activity
Stabilization
Water
Enzymes
phenyliminoribitol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase. / Parkin, David W.; Schramm, Vern L.

In: Biochemistry, Vol. 34, No. 42, 1995, p. 13961-13966.

Research output: Contribution to journalArticle

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abstract = "The transition-state structure for inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupi{\~n}{\'a}n, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitors with observed Km/Ki values up to 2 × 105 [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262]. Although slow-onset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The Km value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6-10.5. Dependencies of Vmax and Vmax/Km on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1-(S)-Phenyl-1,4-dideoxy-1,4-imino-D-ribitol (phenyliminoribitol) binds with an equilibrium Kd of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the Vmax profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding. It has been proposed that the positive charge of protonated inhibitor (pK 6.5) is a recognition feature for binding as a transition-state inhibitor. However, the pH analysis indicates that the neutral inhibitor is the preferred species for binding the active form of the enzyme. The slow-onset phase of phenyliminoribitol binding disappears at low pH, suggesting that a time-dependent protonation of the bound complex could be responsible for the slow-onset phase of inhibition.",
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