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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. [ABSTRACT FROM AUTHOR]

The nucleoside hydrolases (NHs) are a family of nucleoside-modifying enzymes. They play an important role in the purine-salvage pathway of many pathogenic organisms which are unable to synthesize purines de novo. Although well characterized in protozoan parasites, their precise function and mechanism remain unclear in other species. For the first time, NHs from Caenorhabditis elegans and Campylobacter jejuni, which are representatives of mesozoa and bacteria, respectively, have been cloned and purified. Steady-state kinetics indicate a different substrate-specificity profile to previously described hydrolases. Native diffraction data sets were collected from crystals of NH from each organism. The hexagonal crystals (space group P6[SUB2]22 or P6[SUB4]22) of NH from C. elegans diffracted to a resolution of 2.8 Å, while the data set from the orthorhombic crystals (space group I222 or I2[SUB1]2[SUB1]2[SUB1]) of NH from C. jejuni could be processed to 1.7 Å resolution. The unit-cell parameters were a = b = 102.23, c = 117.27 Å in the former case and a = 110.13, b = 100.13, c = 81.37 Å in the latter. [ABSTRACT FROM AUTHOR]