View source for Phosphoenolpyruvate carboxylase

{{Short description|Class of enzymes}}
{{infobox enzyme
| Name       = Phosphoenolpyruvate carboxylase
| EC_number  = 4.1.1.31
| CAS_number = 9067-77-0
| GO_code    = 0008964
| image      = PEP Carboxylase Structure.png
| width      =
| caption    = the Phosphoenolpyruvate (PEP) carboxylase single subunit structure (generated by PyMOL)]
}}
{{Pfam_box
| Symbol = PEPcase
| Name = Phosphoenolpyruvate carboxylase
| image =
| width =
| caption =
| Pfam= PF00311
| InterPro= IPR001449
| SMART=
| Prosite = PDOC00330
| SCOP = 1fiy
| TCDB =
| OPM family=
| OPM protein=
| PDB= 3ZGE   
}}

'''Phosphoenolpyruvate carboxylase''' (also known as '''PEP carboxylase''', '''PEPCase''', or '''PEPC'''; {{EnzExplorer|4.1.1.31}}, PDB ID: 3ZGE) is an [[enzyme]] in the family of [[carboxy-lyases]] found in plants and some bacteria that catalyzes the addition of [[bicarbonate]] (HCO<sub>3</sub><sup>−</sup>) to [[phosphoenolpyruvate]] (PEP) to form the four-carbon compound [[oxaloacetate]] and inorganic [[phosphate]]:<ref name="KaiMatsumura2003">{{cite journal | vauthors = Kai Y, Matsumura H, Izui K | title = Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms | journal = Archives of Biochemistry and Biophysics | volume = 414 | issue = 2 | pages = 170–9 | date = June 2003 | pmid = 12781768 | doi = 10.1016/S0003-9861(03)00170-X }}</ref>

:PEP + HCO<sub>3</sub><sup>−</sup> → oxaloacetate + Pi

This reaction is used for [[carbon fixation]] in [[Crassulacean acid metabolism|CAM]] (crassulacean acid metabolism) and [[C4 carbon fixation|{{C4}}]] organisms, as well as to regulate [[flux (metabolism)|flux]] through the [[citric acid cycle]] (also known as [[citric acid cycle|Krebs]] or [[citric acid cycle|TCA]] cycle) in bacteria and plants. The enzyme structure and its two step catalytic, irreversible mechanism have been well studied. PEP carboxylase is highly regulated, both by [[phosphorylation]] and [[allosteric regulation|allostery]].

==Enzyme structure==
The PEP carboxylase enzyme is present in plants and some types of bacteria, but not in fungi or animals (including humans).<ref name="CholletVidal1996">{{cite journal | vauthors = Chollet R, Vidal J, O'Leary MH | title = Phosphoenolpyruvate Carboxylase: A Ubiquitous, Highly Regulated Enzyme in Plants | journal = Annual Review of Plant Physiology and Plant Molecular Biology | volume = 47 | issue = 1 | pages = 273–298 | date = June 1996 | pmid = 15012290 | doi = 10.1146/annurev.arplant.47.1.273 | s2cid = 28888382 | url = http://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1041&context=plantscifacpub | url-access = subscription }}</ref> The genes vary between organisms, but are strictly [[conserved sequence|conserved]] around the [[active site|active]] and [[allosteric regulation|allosteric sites]] discussed in the mechanism and regulation sections. [[biomolecular structure|Tertiary structure]] of the enzyme is also conserved.<ref name="PaulusSchlieper2013">{{cite journal | vauthors = Paulus JK, Schlieper D, Groth G | title = Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution | journal = Nature Communications | volume = 4 | issue = 2 | pages = 1518 | year = 2013 | pmid = 23443546 | pmc = 3586729 | doi = 10.1038/ncomms2504 }}</ref>

The crystal structure of PEP carboxylase in multiple organisms, including [[maize|''Zea mays'']] (maize), and ''[[Escherichia coli]]'' has been determined.<ref name="PaulusSchlieper2013"/> The overall enzyme exists as a dimer-of-dimers: two identical subunits closely interact to form a dimer through salt bridges between [[arginine]] (R438 - exact positions may vary depending on the origin of the gene) and [[glutamic acid]] (E433) residues.<ref name="KaiMatsumura1999">{{cite journal | vauthors = Kai Y, Matsumura H, Inoue T, Terada K, Nagara Y, Yoshinaga T, Kihara A, Tsumura K, Izui K | title = Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 3 | pages = 823–8 | date = February 1999 | pmid = 9927652 | doi = 10.1073/pnas.96.3.823 | pmc = 15309 | doi-access = free }}</ref> This dimer assembles (more loosely) with another of its kind to form the four subunit complex. The monomer subunits are mainly composed of [[alpha helix|alpha helices]] (65%),<ref name="KaiMatsumura2003"/> and have a mass of 106kDa each.<ref name="GonzalezIglesias1986">{{cite journal | vauthors = Gonzalez DH, Iglesias AA, Andreo CS | title = Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate | journal = Archives of Biochemistry and Biophysics | volume = 245 | issue = 1 | pages = 179–86 | date = February 1986 | pmid = 3947097 | doi = 10.1016/0003-9861(86)90203-1 }}</ref> The sequence length is about 966 [[amino acid]]s.<ref>{{PDB|3ZGE}}; {{cite journal | vauthors = Paulus JK, Schlieper D, Groth G | title = Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution | journal = Nature Communications | volume = 4 | pages = 1518 | date = 19 April 2018 | pmid = 23443546 | doi = 10.1038/ncomms2504 | pmc = 3586729 }}</ref>

The enzyme active site is not completely characterized. It includes a conserved [[aspartic acid]] (D564) and a [[glutamic acid]] (E566) residue that non-covalently bind a divalent metal [[cofactor (biochemistry)|cofactor]] ion through the [[carboxylic acid]] functional groups.<ref name="KaiMatsumura2003"/> This metal ion can be [[magnesium]], [[manganese]] or [[cobalt]] depending on the organism,<ref name="KaiMatsumura2003"/><ref name="CholletVidal1996"/> and its role is to coordinate the phosphoenolpyruvate molecule as well as the reaction intermediates. A [[histidine]] (H138) residue at the active site is believed to facilitate proton transfer during the catalytic mechanism.<ref name="KaiMatsumura2003"/><ref name="KaiMatsumura1999"/>

==Enzyme mechanism==
The mechanism of PEP carboxylase has been well studied. The enzymatic mechanism of forming [[oxaloacetate]] is very [[exergonic]], and thereby irreversible, in biochemical standard conditions; the biological standard [[Gibbs free energy]] change (∆G°’) is −30&nbsp;kJ⋅mol<sup>−1</sup>.<ref name="KaiMatsumura2003"/> The [[substrate (biology)|substrates]] and cofactor bind in the following order: metal cofactor (either Co<sup>2+</sup>, Mg<sup>2+</sup>, or Mn<sup>2+</sup>), PEP, [[bicarbonate]] (HCO<sub>3</sub><sup>−</sup>).<ref name="KaiMatsumura2003"/><ref name="CholletVidal1996"/> The mechanism proceeds in two major steps, as described below and shown in figure 2:
[[File:PEP Carboxylase Mechanism.png|thumb|400px|left|Figure 2: the Phosphoenolpyruvate (PEP) carboxylase enzymatic mechanism converting bicarbonate and PEP to oxaloacetate and phosphate.]]

# The bicarbonate acts as a [[nucleophile]] to attack the [[phosphate]] group in PEP. This results in the splitting of PEP into a carboxyphosphate and the (very reactive) [[enol]]ate form of [[pyruvate]].
# Proton transfer takes place at the carboxyphosphate. This is most likely modulated by a [[histidine]] (H138) residue that first deprotonates the carboxy side, and then, as an acid, protonates the phosphate part.<ref name="KaiMatsumura2003"/> The carboxyphosphate then exothermically decomposes into [[carbon dioxide]] and inorganic phosphate, at this point making this an irreversible reaction. Finally, after the decomposition, the carbon dioxide is attacked by the enolate to form oxaloacetate.<ref name="KaiMatsumura2003"/><ref name="CholletVidal1996"/><ref name="FujitaIzui1984">{{cite journal | vauthors = Fujita N, Izui K, Nishino T, Katsuki H | title = Reaction mechanism of phosphoenolpyruvate carboxylase. Bicarbonate-dependent dephosphorylation of phosphoenol-alpha-ketobutyrate | journal = Biochemistry | volume = 23 | issue = 8 | pages = 1774–9 | date = April 1984 | pmid = 6326809 | doi = 10.1021/bi00303a029 }}</ref>

The metal cofactor is necessary to coordinate the enolate and carbon dioxide intermediates; the CO<sub>2</sub> molecule is only lost 3% of the time.<ref name="CholletVidal1996"/> The active site is [[hydrophobe|hydrophobic]] to exclude [[water]], since the carboxyphosphate intermediate is susceptible to [[hydrolysis]].<ref name="KaiMatsumura2003"/>
{{clear}}

== Function ==
The three most important roles that PEP carboxylase plays in plants and bacteria metabolism are in the [[C4 carbon fixation|{{C4}} cycle]], the [[Crassulacean acid metabolism|CAM cycle]], and the [[citric acid cycle]] biosynthesis flux.

The primary mechanism of carbon dioxide assimilation in plants is through the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (also known as [[RuBisCO]]), that adds CO<sub>2</sub> to [[ribulose-1,5-bisphosphate]] (a 5 carbon sugar), to form two molecules of [[3-phosphoglycerate]] (2x3 carbon sugars). However, at higher temperatures and lower CO<sub>2</sub> concentrations, RuBisCO adds [[oxygen]] instead of carbon dioxide, to form the unusable product [[glycolate]] in a process called [[photorespiration]]. To prevent this wasteful process, plants increase the local CO<sub>2</sub> concentration in a process called the [[C4 carbon fixation|{{C4}} cycle]].<ref name="PaulusSchlieper2013"/><ref name="Leegood2007">{{cite journal | vauthors = Leegood RC | title = A welcome diversion from photorespiration | journal = Nature Biotechnology | volume = 25 | issue = 5 | pages = 539–40 | date = May 2007 | pmid = 17483837 | doi = 10.1038/nbt0507-539 | s2cid = 5015366 }}</ref> PEP carboxylase plays the key role of binding CO<sub>2</sub> in the form of [[bicarbonate]] with PEP to create oxaloacetate in the [[mesophyll tissue]]. This is then converted back to [[pyruvate]] (through a [[malate]] intermediate), to release the CO<sub>2</sub> in the deeper layer of [[bundle sheath cells]] for carbon fixation by [[RuBisCO]] and the [[Calvin cycle]]. Pyruvate is converted back to PEP in the mesophyll cells, and the cycle begins again, thus actively pumping CO<sub>2</sub>.<ref name="CholletVidal1996"/><ref name="Hatch2002">{{cite journal | vauthors = Hatch MD | title = C(4) photosynthesis: discovery and resolution | journal = Photosynthesis Research | volume = 73 | issue = 1–3 | pages = 251–6 | year = 2002 | pmid = 16245128 | doi = 10.1023/A:1020471718805 | s2cid = 343310 }}</ref><ref name="KeeleyRundel2003">{{cite journal|last1=Keeley|first1=Jon E.|last2=Rundel|first2=Philip W. | name-list-style = vanc |title=Evolution of CAM and C4Carbon‐Concentrating Mechanisms|journal=International Journal of Plant Sciences|volume=164|issue=S3|year=2003|pages=S55–S77 |doi=10.1086/374192|s2cid=85186850}}</ref>

The second important and very similar biological significance of PEP carboxylase is in the [[Crassulacean acid metabolism|CAM cycle]]. This cycle is common in organisms living in arid habitats. Plants cannot afford to open [[stomata]] during the day to take in CO<sub>2</sub>, as they would lose too much water by [[transpiration]]. Instead, stomata open at night, when water evaporation is minimal, and take in CO<sub>2</sub> by fixing with PEP to form [[oxaloacetate]] though PEP carboxylase. Oxaloacetate is converted to [[malate]] by [[malate dehydrogenase]], and stored for use during the day when the [[light dependent reaction]] generates energy (mainly in the form of [[Adenosine triphosphate|ATP]]) and [[reducing equivalents]] such as [[NADPH]] to run the [[Calvin cycle]].<ref name="CholletVidal1996"/><ref name="PaulusSchlieper2013"/><ref name="KeeleyRundel2003"/>

Third, PEP carboxylase is significant in non-photosynthetic metabolic pathways. Figure 3 shows this metabolic flow (and its regulation). Similar to [[pyruvate carboxylase]], PEP carboxylase replenishes oxaloacetate in the citric acid cycle. At the end of [[glycolysis]], PEP is converted to [[pyruvate]], which is converted to acetyl-coenzyme-A ([[acetyl-CoA]]), which enters the citric acid cycle by reacting with oxaloacetate to form [[citrate]]. To increase flux through the cycle, some of the PEP is converted to oxaloacetate by PEP carboxylase. Since the citric acid cycle intermediates provide a hub for metabolism, increasing flux is important for the [[biosynthesis]] of many molecules, such as for example [[amino acids]].<ref name="CousinsBaroli2007">{{cite journal | vauthors = Cousins AB, Baroli I, Badger MR, Ivakov A, Lea PJ, Leegood RC, von Caemmerer S | title = The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance | journal = Plant Physiology | volume = 145 | issue = 3 | pages = 1006–17 | date = November 2007 | pmid = 17827274 | pmc = 2048775 | doi = 10.1104/pp.107.103390 }}</ref>

==Regulation==
[[File:PEP Carboxylase Regulation.png|thumb|500px|Figure 3: the Phosphoenolpyruvate (PEP) carboxylase regulation pathways]]

PEP carboxylase is mainly subject to two levels of regulation: [[phosphorylation]] and [[allosteric regulation|allostery]]. Figure 3 shows a schematic of the regulatory mechanism.

[[Phosphorylation]] by phosphoenolpyruvate carboxylase [[kinase]] turns the enzyme on, whereas phosphoenolpyruvate carboxylase [[phosphatase]] turns it back off. Both kinase and phosphatase are regulated by [[transcription (genetics)|transcription]]. It is further believed that [[malate]] acts as a feedback [[Enzyme inhibitor|inhibitor]] of kinase expression levels, and as an activator for phosphatase expression (transcription).<ref name="Nimmo2000">{{cite journal | vauthors = Nimmo HG | title = The regulation of phosphoenolpyruvate carboxylase in CAM plants | journal = Trends in Plant Science | volume = 5 | issue = 2 | pages = 75–80 | date = February 2000 | pmid = 10664617 | doi = 10.1016/S1360-1385(99)01543-5 }}</ref> Since oxaloacetate is converted to malate in CAM and {{C4}} organisms, high concentrations of malate activate phosphatase expression - the phosphatase subsequently de-phosphorylates and thus de-actives PEP carboxylase, leading to no further accumulation of oxaloacetate and thus no further conversion of oxaloacetate to malate. Hence malate production is down-regulated.<ref name="KaiMatsumura2003"/><ref name="Nimmo2000"/>

The main [[allosteric regulation|allosteric inhibitors]] of PEP carboxylase are the [[carboxylic acids]] [[malate]] (weak) and [[aspartate]] (strong).<ref name="GonzalezIglesias1986"/><ref name="Nimmo2000"/> Since malate is formed in the next step of the CAM and {{C4}} cycles after PEP carboxylase catalyses the condensation of CO<sub>2</sub> and PEP to oxaloacetate, this works as a feedback inhibition pathway. Oxaloacetate and aspartate are easily inter-convertible through a [[transaminase]] mechanism; thus high concentrations of aspartate are also a pathway of feedback inhibition of PEP carboxylase.

The main allosteric activators of PEP carboxylase are [[acetyl-CoA]]<ref name="Morikawa1979">{{cite journal | vauthors = Morikawa M, Izui K, Taguchi M, Katsuki H | title = Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. Estimation of the activities in the cells grown on various compounds | journal = Journal of Biochemistry | volume = 87 | issue = 2 | pages = 441–9 | date = February 1980 | pmid = 6987214 | doi =  10.1093/oxfordjournals.jbchem.a132764}}</ref> and [[fructose-1,6-bisphosphate]] (F-1,6-BP).<ref name="KaiMatsumura2003"/><ref name="Morikawa1979"/> Both molecules are indicators of increased [[glycolysis]] levels, and thus positive feed-forward [[effector (biology)|effectors]] of PEP carboxylase. They signal the need to produce oxaloacetate to allow more flux through the [[citric acid cycle]]. Additionally, increased [[glycolysis]] means a higher supply of PEP is available, and thus more storage capacity for binding CO<sub>2</sub> in transport to the [[Calvin cycle]]. It is also noteworthy that the negative [[effector (biology)|effectors]] [[aspartate]] competes with the positive effector [[acetyl-CoA]], suggesting that they share an allosteric binding site.<ref name="Smith1970">{{cite journal | vauthors = Smith TE | title = Escherichia coli phosphoenolpyruvate carboxylase: competitive regulation by acetyl-coenzyme A and aspartate | journal = Archives of Biochemistry and Biophysics | volume = 137 | issue = 2 | pages = 512–22 | date = April 1970 | pmid = 4909168 | doi = 10.1016/0003-9861(70)90469-8 }}</ref>

Studies have shown that energy equivalents such as [[Adenosine monophosphate|AMP]], [[Adenosine diphosphate|ADP]] and [[Adenosine triphosphate|ATP]] have no significant effect on PEP carboxylase.<ref name="CoombsMaw1974">{{cite journal | vauthors = Coombs J, Maw SL, Baldry CW | title = Metabolic regulation in C4 photosynthesis: PEP-carboxylase and energy charge | journal = Planta | volume = 117 | issue = 4 | pages = 279–92 | date = December 1974 | pmid = 24458459 | doi = 10.1007/BF00388023 | s2cid = 25003418 }}</ref>

The magnitudes of the allosteric effects of these different molecules on PEP carboxylase activity depend on individual organisms.<ref name="SchullerPlaxton1990">{{cite journal | vauthors = Schuller KA, Plaxton WC, Turpin DH | title = Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum: Properties Associated with Replenishment of Tricarboxylic Acid Cycle Intermediates during Ammonium Assimilation | journal = Plant Physiology | volume = 93 | issue = 4 | pages = 1303–11 | date = August 1990 | pmid = 16667617 | pmc = 1062672 | doi = 10.1104/pp.93.4.1303 }}</ref>

== References ==
{{Reflist}}

{{Carbon-carbon lyases}}
{{Enzymes}}
{{Portal bar|Biology|border=no}}

[[Category:EC 4.1.1]]
[[Category:Photosynthesis]]