A cis-proline to alanine mutant of E. coli aspartate transcarbamoylase: Kinetic studies and three-dimensional crystal structures

The only cis-proline residue in Escherichia coli aspartate transcarbamoylas e has been replaced by alanine using site-specific mutagenesis. The Pro268- ->Ala enzyme exhibits a 40-fold reduction in enzyme activity and decreased substrate affinity toward carbamoyl phosphate and aspartate compared to the corresponding values for the wild-type enzyme. The concentration of the bi substrate analogue N-phosphonacetyl-L-aspartate (PALA) required to activate the mutant enzyme to the same extent as the wild-type enzyme is significan tly increased. The heterotropic effects of ATP and CTP upon the Pro268-->Al a enzyme are also altered. Crystal structures of the Pro268-->Ala enzyme in both T- and R-states show that the cis-peptidyl linkage between Leu267 and Ala268 is maintained. However, the tertiary structure of both the catalyti c and regulatory chains has been altered by the amino acid substitution, an d the mobility of the active-site residues is increased for the R-state str ucture of Pro268-->Ala enzyme as comparison with the wild-type R-state stru cture. These structural changes are responsible for the loss of enzyme acti vity. Thus, Pro268 is required for the proper positioning of catalytically critical residues in the active site and is important for the formation of the high-activity high-affinity R-state of E. coli aspartate transcarbamoyl ase.