A SINGLE MUTATION IN THE REGULATORY CHAIN OF ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE RESULTS IN AN EXTREME T-STATE STRUCTURE

Kinetic analysis of a mutant version of Escherichia coli aspartate tra nscarbamoylase in which Thr82 in the regulatory chain (Thr82r) was rep laced by Ala results in a shift in the T reversible arrow R equilibriu m towards the T-state. In order to understand the structural determina nts of this T-state stabilization, the X-ray structure of the unligand ed Thr82r --> Ala enzyme was determined at 2.6 Angstrom resolution and refined to a crystallographic residual of 0.175. The structure of the mutant r1 regulatory chain is more similar to that of the r6 regulato ry chain than observed for the wild-type enzyme, resulting in a more s ymmetric structure. Furthermore, the structural changes in the mutant enzyme appears to occur only in the r1 chain, while the r6 chain is al most identical in structure to that of the r6 chain of the wild-type e nzyme. The structure of the mutant enzyme exhibits alterations in the subunit interfaces between the regulatory and catalytic chains, as wel l as in the interface between the allosteric and zinc domains within t he regulatory chain. Moreover, the regulatory dimers are rotated aroun d their respective 2-fold axes approximately 1 degrees beyond the rota tion which occurs in the wild-type T-state enzyme. The structural anal ysis indicates that the enzyme is an ''extreme'' T-state, in which a l arger rotation of the regulatory dimers is required for the T to R tra nsition compared to the wild-type enzyme. This extreme T-state structu re correlates well with the kinetic parameters determined for the muta nt enzyme, showing a stabilized T-state. Furthermore, the structural a nalysis of the mutant enzyme suggests that replacement of Thr82r with Ala alters the local conformation of the nucleotide binding pocket and therefore offers a plausible explanation for the reduced affinity of the enzyme for nucleotides. (C) 1998 Academic Press