A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant.
Escherichia coli asparaginase II catalyzes the hydrolysis of L-asparagine to L-aspartate via a threonine-bound acyl-enzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.
Palm
G J
GJ
Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA.
Lubkowski
J
J
Derst
C
C
Schleper
S
S
Röhm
K H
KH
Wlodawer
A
A
D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation
D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D001215:Asparaginase