Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase.
Periplasmic Escherichia coli L-asparaginase II with Y25F mutation in the active-site cavity has been obtained by recombinant techniques. The protein was crystallized in a new hexagonal form (P6(5)22). Single crystals of this polymorph, suitable for X-ray diffraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipitant (pH 4.8). The crystals are characterized by a = 81.0, c = 341.1 A and diffract to 2.45 A resolution. The asymmetric unit contains two protein molecules arranged into an AB dimer. The physiologically relevant ABA'B' homotetramer is generated by the action of the crystallographic 2-fold axis along [1, -1, 0]. Kinetic studies show that the loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs kcat without significantly affecting Km.
Kozak
M
M
Department of Macromolecular Physics, Faculty of Physics, A. Mickiewicz University, Poznań, Poland. mkozak@main.amu.edu.pl
Jaskólski
M
M
Röhm
K H
KH
D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D001215:Asparaginase