Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase.

M Kozak; M Jaskólski; K H Röhm

doi:10.18388/abp.2000_3998

Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase.

M Kozak Department of Macromolecular Physics, Faculty of Physics, A. Mickiewicz University, Poznań, Poland. mkozak@main.amu.edu.pl;
M Jaskólski
K H Röhm

DOI: https://doi.org/10.18388/abp.2000_3998

Abstract

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.

Published

2000-09-30

Issue

Vol. 47 No. 3 (2000)

Section

Articles

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