Monitoring the transition from the T to the R state in E.coli aspartate transcarbamoylase by X-ray crystallography: crystal structures of the E50A mutant enzyme in four distinct allosteric states.

A detailed description of the transition that allosteric enzymes undergo constitutes a major challenge in structural biology. We have succeeded in trapping four distinct allosteric states of a mutant enzyme of Escherichia coli aspartate transcarbomylase and determining their structures by X-ray crystallography. The mutant version of aspartate transcarbamoylase in which Glu50 in the catalytic chains was replaced by Ala destabilizes the native R state and shifts the equilibrium towards the T state. This behavior allowed the use of substrate analogs such as phosphonoacetamide and malonate to trap the enzyme in T-like and R-like structures that are distinct from the T-state structure of the wild-type enzyme (as represented by the structure of the enzyme with CTP bound and the R-state structure as represented by the structure with N-(phosphonacetyl)-L-aspartate bound). These structures shed light on the nature and the order of internal structural rearrangements during the transition from the T to the R state. They also suggest an explanation for diminished activity of the E50A enzyme and for the change in reaction mechanism from ordered to random for this mutant enzyme.