Pre-Steady-State Analysis of the Nucleoside Hydrolase of <i>Trypanosoma vivax</i>. Evidence for Half-of-the-Sites Reactivity and Rate-Limiting Product Release

Vandemeulebroucke, An; Versées, Wim; Vos, S. De; Holsbeke, Els Van; Steyaert, Jan

doi:10.1021/bi0347914

Cited by 22 publications

(40 citation statements)

References 23 publications

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“…In the next step, ribose binding to the R252A mutant was analyzed by stopped flow fluorescence. The concentration dependence of the observed rates of ribose binding shows that this process occurs via the same two-step binding mechanism as seen for the wild type enzyme (23). This twostep binding mechanism consists of a first collision step (K ribose in Scheme 1) to form a loosely bound enzyme ribose complex, followed by an isomerization that converts this loosely bound complex into a tightly bound complex (k 4 and k Ϫ4 in Scheme 1).…”

Section: Proline Scan Of the Flexible Loop 2-it Had Previously Been Smentioning

confidence: 75%

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

Vandemeulebroucke¹,

Vos²,

Holsbeke

et al. 2008

Journal of Biological Chemistry

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The nucleoside hydrolase of Trypanosoma vivax hydrolyzes the N-glycosidic bond of purine nucleosides. Structural and kinetic studies on this enzyme have suggested a catalytic role for a flexible loop in the vicinity of the active sites. Here we present the analysis of the role of this flexible loop via the combination of a proline scan of the loop, loop deletion mutagenesis, steady state and pre-steady state analysis, and x-ray crystallography. Our analysis reveals that this loop has an important role in leaving group activation and product release. The catalytic role involves the entire loop and could only be perturbed by deletion of the entire loop and not by single site mutagenesis. We present evidence that the loop closes over the active site during catalysis, thereby ordering a water channel that is involved in leaving group activation. Once chemistry has taken place, the loop dynamics determine the rate of product release.The importance of conformational changes in enzyme catalysis has long been appreciated. These changes are thought to fulfill a number of roles in catalysis: enhanced binding of substrate, correct orientation of catalytic groups, removal of water from the active site, and trapping of intermediates. Loop motion is classified as an enzyme conformational change, where flexible surface loops move to different conformations (1).Flexible loops are also a common feature of the family of nucleoside hydrolases (EC 3.2.2.1) (2). All structures of the nucleoside hydrolases determined until now show two flexible loops in the vicinity of the active sites, which undergo substantial conformational changes upon association of substrates and inhibitors (2). Nucleoside hydrolases (NHs) 4 catalyze the hydrolysis of the N-glycosidic bond of nucleosides, forming ribose and the corresponding base ( Fig. 1) (2). It has been shown that the NH-catalyzed hydrolysis proceeds via a highly dissociative S N 2-type mechanism with an oxocarbenium ion-like transition state (3, 4). A general catalytic mechanism based on three strategies has been proposed: activation of the nucleophilic water molecule, steric and electrostatic stabilization of the oxocarbenium ion, and leaving group (LG) activation by protonation or hydrogen bonding (2).The purine-specific nucleoside hydrolase of Trypanosoma vivax (TvNH) has been very well characterized kinetically and structurally. This allowed us to put forward a detailed catalytic mechanism to accomplish these strategies. An aspartate (Asp 10 ) in the ribose binding pocket acts as a general base to activate the nucleophile water (5). The oxocarbenium ion is stabilized through interactions between the enzyme and all of the hydroxyl groups (6). The third catalytic strategy, LG activation, was thought to be accomplished by a Brönsted acid, which protonates nitrogen-7 (N-7) of the purine base (2, 7-9). However, in TvNH, no obvious proton donor was found. Here, the LG of the substrate is stacked between the two indole side chains of Trp 83 and Trp 260 ( Fig. 2A). A novel catalytic mechanism...

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Section: Proline Scan Of the Flexible Loop 2-it Had Previously Been Smentioning

confidence: 75%

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

Vandemeulebroucke¹,

Vos²,

Holsbeke

et al. 2008

Journal of Biological Chemistry

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“…Due to the complex mechanism of this enzyme, involving rate limiting ribose release and half-of-the-sites reactivity, 8 we performed steady state and pre-steady state kinetic measurements to reveal the microscopic rate constant of bond cleavage (k 2 ; Table 1). The Trp83Ala mutation increases the value of K M for guanosine by three orders of magnitude, but has a negligible effect on k 2 (data not shown) excluding an important catalytic role for this residue.…”

Section: Site-directed Mutagenesis Reveals a Role For Trp260 In Leavimentioning

confidence: 99%

“…6,8 Nucleoside hydrolysis was monitored by the decrease in absorbance, caused by the spectral difference between a nucleoside and the corresponding base. The traces showing burst kinetics were conveniently fitted to the standard equation that accounts for a fast irreversible step ðk 2 Þ followed by slow product release ðk 3 Þ: 36 The kinetics of ribose binding was measured by following the fluorescence quenching upon complex formation at different sugar concentrations.…”

Section: Site-directed Mutagenesis Reveals a Role For Trp260 In Leavimentioning

confidence: 99%

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Leaving Group Activation by Aromatic Stacking: An Alternative to General Acid Catalysis

Versées

Loverix

Vandemeulebroucke

et al. 2004

Journal of Molecular Biology

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108

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General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.

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“…Similar to protonation, N7-methylation of guanosine induces a positive charge in the purine ring, facilitating leaving group departure. Because TvNH follows a complex mechanism with rate-limiting ribose release (22), the kinetic analysis was performed on the second order rate constant k cat /K m , which is independent of product release (23) ( Table I). Removal of the 5Ј-OH group in guanosine causes a 6,000-fold reduction in k cat /K m (corresponding to 5.4 kcal/mol).…”

Section: Modulation Of Purine Basicity Via a C8h-o5ј Hydrogenmentioning

confidence: 99%

Substrate-assisted Leaving Group Activation in Enzyme-catalyzed N-Glycosidic Bond Cleavage

Loverix

Geerlings

McNaughton

et al. 2005

Journal of Biological Chemistry

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In enzymatic depurination of nucleosides, the 5-OH group of the ribose moiety of the substrate is often shown to contribute substantially to catalysis. The purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) fixes the 5-OH group in a gauche,trans orientation about the C4-C5 bond, enabling the 5-oxygen to accept an intramolecular hydrogen bond from the C8-atom of the purine leaving group. High level ab initio quantum chemical calculations indicate that this interaction promotes protonation of the purine at N7. Steady state kinetics comprising engineered substrates confirm that a considerable fraction of the catalytic 5-OH effect can be attributed to leaving group activation.In enzyme-catalyzed ribosyl transfer reactions on nucleosides (e.g. hydrolysis, phosphorolysis), the 5Ј-hydroxyl group of the substrate has often been implicated in catalysis (1-4). Inverse 4Ј-3 H and normal 5Ј-3 H kinetic isotope effects are commonly observed, contrary to the non-enzymatic reactions, and have been invoked to account for a strained conformation of the 5Ј-OH group in the transition state (5). However, care must be taken in the interpretation of these kinetic isotope effects because they may be caused by binding rather then catalysis (6). The present study on the purine-specific nucleoside hydrolase (EC 3.2.2.1) of Trypanosoma vivax (TvNH) 1 aims to elucidate the catalytic role of the 5Ј-OH group, which contributes 5.4 kcal/mol to k cat /K m (1).The putative transition state for enzymatic cleavage of Nglycosidic bonds is generally highly dissociative with substantial lengthening of the scissile bond but no bond formation to the incoming nucleophile (5, 7). The transition state has a high ribo-oxocarbenium character with sp 2 hybridization at C1Ј and a C3Ј-exo pucker. In purine nucleosides, departure of the leaving group is facilitated by protonation at N7 prior to reaching the transition state. In the base aspecific 2 NH of Crithidia fasciculata a histidine residue has been identified as the general acid to accomplish this. However, a remarkable feature of the TvNH enzyme is the apparent lack of an acidic group to protonate the leaving purine. In this enzyme, x-ray crystallography and mutagenic scanning analysis did not reveal a suitable general acid candidate (1). Rather, a tryptophan (Trp-260) was found to be the only catalytic residue in the vicinity of the purine leaving group. A previous study combining quantum chemical calculations, mutagenesis, and presteady state kinetics revealed that an aromatic stacking interaction between Trp-260 and the purine ring contributes to catalysis by raising the basicity of the latter (8). Because enhanced basicity facilitates protonation by solvent, it appears that the TvNH enzyme employs specific, rather than general, acid catalysis.Depurination by TvNH is further catalyzed by interactions with the three hydroxyls of the ribose moiety of the substrate, because the 2Ј-, 3Ј-, and 5Ј-deoxy nucleosides are severely impaired in binding and catalysis (1). The inosine bound in t...

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Pre-Steady-State Analysis of the Nucleoside Hydrolase of Trypanosoma vivax. Evidence for Half-of-the-Sites Reactivity and Rate-Limiting Product Release

Cited by 22 publications

References 23 publications

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

A Flexible Loop as a Functional Element in the Catalytic Mechanism of Nucleoside Hydrolase from Trypanosoma vivax

Leaving Group Activation by Aromatic Stacking: An Alternative to General Acid Catalysis

Substrate-assisted Leaving Group Activation in Enzyme-catalyzed N-Glycosidic Bond Cleavage

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