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Abstract
Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2-methyl-2,4-pentanediol. The space group has been determined as P3(1) or P3(2), with one protein trimer of three identical 36.8-kDa subunits in the asymmetric unit. The unit cell dimensions are a = b = 105.1 A and c = 87.8 A. The crystals diffract well to 3-A resolution and are quite resistant to radiation damage. Single crystals have also been grown of a genetically engineered site-specific mutant for which the replacement of an arginine (Arg-57) to a glycine has been shown to not only drastically affect the enzyme activity but also its kinetic mechanism (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). The crystals of the Arg-57----Gly mutant protein are isomorphous to those of the wild type. Crystal soaking experiments using both wild-type and Arg-57----Gly crystals in the presence of various ligands have provided evidence of specific conformational changes upon substrate binding which supports our previous kinetic and spectroscopic observations.
Highlights
Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2methyl-2,4-pentanediol
Because L-arginine is the penultimate product of this cycle, transcarbamoylation of ornithine is a step in the pathway of arginine biosynthesis
The conformational changes related to the action of ornithine transcarbamoylase are of special interest
Summary
Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2methyl-2,4-pentanediol. Only the Arg-57 mutation results in an enzyme which shows no isomerization when carbamoyl phosphate binds. To elucidate the structurarlole of the ligandinduced conformational changes in the mechanism of action of ornithine transcarbamoylase, a high resolution three-dimensional structure of the enzyme is required. We report our initial analysis on the x-ray diffraction properties of single crystals of the enzyme and one of its site-directed point mutants.
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