Three-dimensional structure of phosphoenolpyruvate carboxylase: A proposed mechanism for allosteric inhibition

Abstract

The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has been determined by x-ray diffraction methods at 2.8-Å resolution by using Escherichia coli PEPC complexed with l-aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a “dimer-of-dimers” form with respect to subunit contact, resulting in an overall square arrangement. The contents of α-helices and β-strands are 65% and 5%, respectively. All of the eight β-strands, which are widely dispersed in the primary structure, participate in the formation of a single β-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the β-barrel. The binding site for l-aspartate is located about 20 Å away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, l-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by l-aspartate.