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One of the many interactions important for stabilizing the T state of aspartate
carbamoyltransferase occurs between residues Tyr240 and Asp271 within one
catalytic chain. The functional importance of this polar interaction was
documented by site-directed mutagenesis in which the tyrosine was replaced by a
phenylalanine [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl.
Acad. Sci. U.S.A. 83, 5866-5870]. In the Tyr240----Phe mutant, the aspartate
concentration required to achieve half-maximum velocity is reduced to 4.7 from
11.9 mM for the native enzyme. Here, we report an X-ray crystallographic study
of the Tyr240----Phe enzyme at 2.5-A resolution. While employing crystallization
conditions identical with those used to grow cytidine triphosphate ligated
T-state crystals of the native enzyme, we obtain crystals of the mutant enzyme
that are isomorphous to those of the native enzyme. Refinement of the mutant
structure to an R factor of 0.219 (only eight solvent molecules included) and
subsequent comparison to the native T-state structure indicate that the
quaternary, tertiary, and secondary structures of the mutant are similar to
those for the native T-state enzyme. However, the conformation of Phe240 in one
of the two crystallographically independent catalytic chains contained in the
asymmetric unit is significantly different from the conformation of Tyr240 in
the native T-state enzyme and similar to the conformation of Tyr240 as
determined from the R-state structure [Ke, H.-M., Lipscomb, W. N., Cho, Y. J.,
& Honzatko, R. B. (1988) J. Mol. Biol. (in press)], thereby indicating that
the mutant has made a conformational change toward the R state, localized at the
site of the mutation in one of the catalytic chains.
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