ABSTRACT
The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4. 1.1.31) has been determined by x-ray diffraction methods at 2.8-A 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 alpha-helices and beta-strands are 65% and 5%, respectively. All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-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 beta-barrel. The binding site for L-aspartate is located about 20 A 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.
Subject(s)
Aspartic Acid/pharmacology , Phosphoenolpyruvate Carboxylase/chemistry , Phosphoenolpyruvate Carboxylase/metabolism , Protein Structure, Secondary , Allosteric Regulation , Amino Acid Sequence , Amino Acid Substitution , Arginine , Aspartic Acid/chemistry , Computer Simulation , Conserved Sequence , Crystallography, X-Ray , Cysteine , Dimerization , Escherichia coli/enzymology , Models, Molecular , Molecular Sequence Data , Phosphoenolpyruvate Carboxylase/antagonists & inhibitors , Sequence Alignment , Sequence Homology, Amino Acid , Zea mays/enzymologyABSTRACT
We determined the crystal structure of spinach ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) by x-ray diffraction at 1.8-A resolution and found that the enzyme contained two kinds of S, SI and SII, present in equal number and disposed in an orderly way within the Rubisco holoenzyme. The electron density maps suggested that leucine was at residue 56 in SI, although histidine was at that position in SII. There were other residue differences. Thus, spinach Rubisco has a L8SI4SII4 subunit structure. The orderly disposition of the heterogeneous small subunits in the Rubisco holoenzyme provides accounts of a multigene family of S in plants.
