 |
PDBsum entry 5dnd
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Experimental data in support of a direct displacement mechanism for type i/ii l-Asparaginases.
|
|
|
Authors
|
|
A.M.Schalk,
A.Antansijevic,
M.Caffrey,
A.Lavie.
|
|
|
Ref.
|
|
J Biol Chem, 2016,
291,
5088-5100.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
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 of 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, whereas 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, and 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.
|
|
|
|
|
|
|
