Three-dimensional structure of phospho enol pyruvate 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 ␣-helices and ␤-strands are 65% and 5%, respectively. All of the eight ␤-strands, which are widely dispersed in the primary structure, participate in the formation of a single ␤-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 ␤-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.

Maize Phosphoenolpyruvate Carboxylase

“…The catalytic site could be assigned to be above the C-terminal side of ␤-barrel, consistent with the data of site-directed mutagenesis (19) (Fig. 2A).…”

“…Similarly the S 0.5 values for PEP for the mutant enzymes were not as markedly decreased upon phosphorylation as for the WT enzyme (Table I). Previously, a molecular mechanism for the decrease of allosteric inhibition caused by phosphorylation had been proposed by us on the basis of the three-dimensional structure of E. coli PEPC (19). It was postulated that, when the flexible N-terminal peptide is phosphorylated at Ser-15, the resulting negatively charged region will occupy the inhibitor-binding site where basic residues are clustered.…”

“…Recently, the three-dimensional struc-ture of PEPC was solved by x-ray crystallographic analysis, as reviewed previously (4,18). The structure of PEPC of Escherichia coli (EcPEPC) was thought to be in an inactive state complexed with an allosteric inhibitor, Asp (19). On the other hand, the structure of PEPC for C 4 photosynthesis from Z. mays (ZmPEPC) was thought to be in an activated state, because crystallization was carried out in the presence of ethylene glycol, an activating and compatible solute (20).…”

Nitrogen-Dependent Carbon Fixation by Picoplankton In Culture and in the Mississippi River

“…Moreover, R587 of E. coli PEPC G-rich loop, which corresponds to R683 on the Synechococcus PCC 7002 PEPC, plays a role in inhibition by aspartate. Aspartate binding to this residue traps the loop and prevents it from contributing to the active site (9). Furthermore, E. coli and Synechococcus PCC 7002 contain the aspartate-binding site homology, which is composed of three domains: EM(T/V)(L/F)(S/A)K, LRN(G/I)(T/Y) and MRNTG.…”

“…The enzyme contributes to photosynthetic and anaplerotic CO 2 fixation. The best-described bacterial PEPC is that found in Escherichia coli; its physical and chemical properties have been extensively investigated (16), and its three-dimensional structure has been determined at 2.8 Å resolution (9). E. coli PEPC is a tetrameric protein composed of four identical subunits of approximately 100 KDa each.…”