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The T----R transition of the cooperative enzyme aspartate carbamoyltransferase
occurs at pH 7 in single crystals without visibly cracking many of the crystals
and leaving those uncracked suitable for single-crystal X-ray analysis. To
promote the T----R transition, we employ the competitive inhibitors of carbamoyl
phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate,
respectively. In response to PAM binding to the T-state crystals, residues Thr
53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the
phosphonate and amino group of PAM. These changes induce a conformation that can
bind tightly the aspartate analogue malonate, which thereby effects the
allosteric transition. We prove this by showing that PAM-ligated T-state
crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred
to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to
R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4
A). The R-state structure in which the T----R transition occurs within the
crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state
structure determined at pH 7 in which the crystals were initially grown in a
solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer
containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and
malonate ligated R-state structures are very similar to both the carbamoyl
phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated
structures, even though the R-state structures reported here were determined at
pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for
the three structures.
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