This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. E. coli aspartate transcarbamoylase is a classical example of allosteric enzyme, possessing homotropic and heterotropic cooperativities, which have a fundamental role in feedback regulation in the pyrimidine biosynthesis pathway. We combine x-ray crystallography and solution x-ray solution techniques to study a large scale quaternary structure change associated with the allosteric transition. High resolution structures obtained by crystallography are sometimes biased by the physical constraints of the unit cell, and solution scattering is used to evaluate crystal structures as well as to obtain true physiological structures in solution. Solution x-ray scattering is frequently used to investigate time-course of structural change upon ligand binding. We have recently investigated several mutant versions of ATCase, all containing substitutions of critical amino acid residues within the active site by equilibrium and time-resolved solution x-ray scattering. Two mutant enzymes H134A and R167Q, remained in the T state after addition of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), and two others, R229A and Q231L, were shifted only partly towards the R state. The mutant enzymes that did exhibit a shift towards the R state after addition of the natural substrates (S52A, K84A, Q231L and R296A) all had much slower T ->R transition rates than the wild-type enzyme. Most strikingly the T ->R transition rate for these mutant enzymes after addition of PALA was unchanged as compared to the wild-type enzyme. These data indicate that the loss of a single interaction in the binding site is not enough to eliminate the ability of the enzyme to undergo the T to R transition, unless the residue making that interaction is also involved in an interdomain R-state stabilizing salt link or forms hydrogen bonds with other active site residues. In addition, the data suggest different pathways for the allosteric transition after the binding of PALA as compared to the natural substrate
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