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

Versées, Wim; Loverix, Stefan; Vandemeulebroucke, An; Geerlings, Pau̧l; Steyaert, Jan

doi:10.1016/j.jmb.2004.02.049

Cited by 85 publications

(114 citation statements)

References 37 publications

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“…Although TvNH lacks a general acid, it is equally catalytically proficient to nucleoside hydrolases from other sources (17). We have previously shown that, instead of employing general acid catalysis, TvNH activates the leaving purine base by increasing the N7 basicity (hence favoring its protonation) via a parallel aromatic stacking interaction with the indole side chain of Trp-260 (8,24). The high level ab initio calculations presented here rule out a previously presumed stabilizing catalytic interaction between 5ЈO and 4ЈO.…”

Section: Discussionmentioning

confidence: 62%

“…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.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 62%

mentioning

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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“…The catalytic water is not apparent in earlier reports of the crystal structure of RTA, and Glu-177 or Arg-180 was considered to be a candidate base for activation (32,33). In the RTA-and SAP-cyclic G(9-DA)GA 2Ј-OMe inhibitor complexes, the 9-deazaadenyl bases share a common binding orientation, the 1-aza-sugar groups are aligned in the same plane, and a water molecule occupies a position near N1Ј appropriate for nucleophilic attack (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Stabilization of N7-protonated adenines occurs via backbone carbonyl oxygens (Gly-121 in RTA and Glu-121 in SAP) with N7 proton donation presumably from solvent. Faceto-face aromatic -stacking of active site tyrosines (Tyr-123 and Tyr-80 in RTA, Tyr-123 and Tyr-73 in SAP) with the adenine base also elevates the pK a of the adenine, and thereby facilitates leaving group activation (32). At one end of the -tetra-stack in both enzymes is a guanidine cation (Arg-134 in both RTA and SAP), adding more cation character through these groups.…”

Section: Discussionmentioning

confidence: 99%

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

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“…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).…”

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confidence: 99%

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

Cited by 85 publications

References 37 publications

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

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

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

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

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