Phosphoenolpyruvate carboxylase
Created by: Peter Polomski
Phosphoenolpyruvate carboxylase (1QB4) is an enzyme with a bound a divalent metal ion such as manganese or magnesium in order to allow for activity. Phosphoenolpyruvate carboxylase, commonly known as PEPC is responsible for the carboxylation of phosphoenolpyruvate. This irreversible process converts the phosphoenolpyruvate to oxaloacetate (OAA), and serves as a crucial intermediate step in the metabolism of Escherichia coli and some other C4 plants such as maize and sugar cane. In (CAM) plants, phosphoenolpyruvate carboxylate plays an instrumental role in capturing environmental CO2. (Matsumuraa, 93-95)
The atomic weight of phosphoenolpyruvatge carboxylase is 99364.34 Da and the isoelectric point is 5.52. (Expasy, 1) There are two functionally important ligands to phosphoenolpyruvate Carboxylase, its bound divalent metal ion and an aspartic acid. The primary function of the aspartic acid residue is internal stabilization of the protein, contributing to the tertiary structure. The purpose of the bound metal ion is to bind to the enolate oxygens of phosphoenolypyruvate or to the enolate intermediate (Ausenhus, 6427). This will cause the hydrolysis of phosphoenolpyruvate into oxaloacetate. Previous research has shown that the metal ion will bind to phosphoenolpyruvate carboxylase even in the absence of phosphoenolpyruvate. (Miller, 6030)(Miziorkoa, 378)
The secondary structure of phosphoenolpyruvate carboxylase is a one subunit protein that is comprised of predominantly alpha helices. There is also a tight collection of beta sheets toward the center of the complex, as well as a scattering of 3/10 helices and random coils. The structure reflects the very specific function of the protein as an enzyme that is responsible for the task of hydrolyzing phosphoenolpyruvate using its active site with a bound metal ion. Crystallization of the protein reveals this binding site to be at two critical residues, Glu-506 and Asp-543. The structure reveals that Mn2+ is bound to the side chain oxygens of these conserved residues which allows it bind and activate the enzymatic activity of phosphoenolpyruvate carboxylase. This binding occurs at the C-terminal end of β-5 and β-6 in eight β-strands of the α/β barrel, respectively (Matsumuraa, 93-95). the coordination sphere of the ion is roughly octahedral, and it is presumed that there must be 4 water molecules to complete the coordination, although these instances of H20 were not observed in electron density at the resolution at which the phosphoenolpyruvate carboxylase was observed.
Phosphoenolpyruvate is accepted by the center of the α/β-barrel, at the C-terminal end of the β-strands in phosphoenolpyruvate carboxylase. The clumps of negatively and positively charged residues in this area assure that phosphoenolpyruvate can be held in an electrostatic environment. The mechanism for the enzymatic action of phosphoenolpyruvate is believed to proceed beginning with a nucleophilic attack by bicarbonate to generate carboxyphosphate and the enolate of pyruvate. There is a positively charged electrostatic pocket that is formed by Arg-396, Arg-581 and Arg-713that contributes to the balancing of the negative charges of the phosphate group during the approach of the transition state, making the phosphorus atom more electrophilic and susceptible to nucleophilic attack by bicarbonate. (Westheimer, 1173) This pathway allows for the phosphoryl group of phosphoenolpyruvate to receive a bicarbonate.
There are a few structurally similar proteins that also have this α/β-barrel. Two such are pyruvate kinase (PK) and pyruvate phosphate dikinase (PPDK) (Dali Server, 1). These two proteins have the most structurally similar barrel for enzymatic activity, despite not having significant similarities in primary structure to phosphoenolpyruvate carboxylase. These analogs of tertiary structure find their role in the metabolic pathway of muscle cells in a variety of organisms, each of which has their own slight variation from the comparison proteins being examined. They use a divalent metal ion to create a charged pocket within the α/β-barrel where the enzymatic reaction will occur applying the necessary metabolic transformation to a Phosphoenolpyruvate analog. The physical structures are quite different, despite having functional similarity, and residue comparison in the primary structure of these functional analogs yields very little homology. The sequences and some secondary structure elements are essential for the effective presence of the analogs in whatever organism they are fulfilling their function for instance the pyruvate kinase in homo sapiens (Dali Server, 1).
Archaeal Type Phosphoenolpyruvate Carboxylate is the closest overall match structurally as it has almost identical sequence to phosphoenolpyruvate carboxylase in plants. There are a host of variations of phosphoenolpyruvate carboxylase in different organisms such as C4 and CAM cycle plants that have almost 100% amino acid identity, but outside of these there are a few similar proteins that have a fair amount of sequence homogeneity but do not exhibit similar function in vitro (PSI BLAST, 1). One such comparable protein is Glutathione S-Transferase, a protein found in Pseudomonas putida that is a transfer protein. It does not share a similar function to phosphoenolpyruvate carboxylase, but it does share some similar traits, such as being comprised primarily of alpha helical secondary structure. Glutathione S-Transferase also is significantly smaller than phosphoenolpyruvate carboxylase, most likely to aid it in its function as a transport protein. It has a fairly high E value of .48 according to a Blast search. This is just below the threshold of .5 for matching similar proteins based on sequence (PSI BLAST, 1).
The function of phosphoenolpyruvate carboxylase is to hydrolize phosphoenolpyruvate in a charged pocket that is activated by the presence of a divalent metal ion. It has a variety of functional analogs in other organisms, and proteins that have reasonably similar sequences and share some secondary structure elements. Ultimately phosphoenolpyruvate carboxylase is a simpler protein that has very specific and important functions for a variety of organisms, and many organisms contain functional analogs that are also crucial in the action of muscles. As an intermediate in the metabolic process of Escherichia coli it exhibits necessary enzymatic activity for the organism to survive.