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

Abstract

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...

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface

“…The enzyme donates several basic groups to counter that charge (20), and dehydration of the active site cleft would strengthen the charge-charge interactions between enzyme and substrates. This is a fundamental mechanistic basis for lid closure over active sites and can yield rate enhancements of orders of magnitude (27)(28)(29). In support of this mechanism, we found that the activity of the catalytic fragment, CCT-236, with its open active site, was much more sensitive to inhibition by raising the ionic strength of the medium than was membrane-bound CCT.…”

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

“…Indeed, the flexible nature of a catalytic loop plays an important role in the activity of a nucleotide hydrolase from Trypanosoma vivax [41]. The flexibility of a loop in E .…”

Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions

“…Alanine was chosen because it represents a deletion of the side chain at the β-carbon. In addition to alanine, many other amino acids, including arginine ( Nanevicz et al 1995 ), cysteine ( Kanaya et al 1990 ), glycine ( Valbuena et al 2003 ), methionine ( Woods et al 1996 ), phenylalanine ( Borngräber et al 1999 ), proline ( Vandemeulebroucke et al 2008 ), and tryptophan ( Zhang et al 2007 ), have been used for scanning mutagenesis, often with a specific hypothesis in mind ( e.g. , that bulky amino acids are important).…”