5dnd

Crystal structure of the Asn-bound guinea pig L-asparaginase 1 catalytic domain active site mutant T116A

spinqualitylabelsall models 5dnd, resolution 2.29Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5dnd is a 4 chain structure with sequence from Cavia porcellus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.29Å Ligands: ASN, EDO Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function H0W0T5_CAVPO Publication Abstract from PubMed Bacterial L-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig L-asparaginase (gpASNase1) as a potential replacement for the immunogenic bacterial enzymes. The exact mechanism used by L-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism while recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A-T116A, K188M, Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for L-asparaginases. Experimental Data in Support of a Direct Displacement Mechanism for Type I/II L-Asparaginases.,Schalk AM, Antanasijevic A, Caffrey M, Lavie A J Biol Chem. 2016 Jan 5. pii: jbc.M115.699884. PMID:26733195 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Asparaginase 3D structures

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