Propionyl-CoA carboxylase
Encyclopedia
Propionyl-CoA carboxylase catalyses the carboxylation
reaction of propionyl CoA in the mitochondrial matrix
. The enzyme is biotin
dependent. The product of the reaction is (S)-methylmalonyl CoA. Propionyl CoA is the end product of metabolism of odd-chain fatty acids, and is also a metabolite of most methyl-branched fatty acids. It is also the main metabolite of valine, and together with acetyl-CoA
, is a metabolite of isoleucine, as well as a methionine
metabolite. Propionyl-CoA
is thus of great importance as a glucose
precursor. (S)-Methylmalonyl-CoA is not directly utilizable by animals; it is acted on by a racemase to give (R)-methylmalonyl-CoA. The latter is converted by methylmalonyl-CoA mutase (one of a very few Vitamin B12 dependent enzymes) to give succinyl-CoA
. The latter is converted to oxaloacetate and then malate
in the Krebs cycle. Export of malate
into the cytosol leads to formation of oxaloacetate, phosphoenol pyruvate, and other gluconeogenic intermediates.
It has been classified both as a ligase
and a lyase
.
The alpha subunit of PCC contains the biotin carboxylase
(BC) and biotin
carboxyl carrier protein (BCCP) domains. A domain known as the BT domain is also located on the alpha subunit and is essential for interactions with the beta subunit. The 8-stranded anti-parallel beta barrel
fold of this domain is particularly interesting. The beta subunit contains the carboxyltransferase (CT) activity.
The BC and CT sites are approximately 55 Å apart, indicative of the entire BCCP domain translocating during catalysis of the carboxylation
of propionyl-CoA
. This provides clear evidence of crucial dimeric interaction between alpha and beta subunits.
The biotin-binding pocket of PCC is hydrophobic and highly conserved. Biotin and propionyl-CoA bind perpendicular to each other in the oxyanion hole containing active site. The native enzyme to biotin ratio has been determined to be one mole native enzyme to 4 moles biotin. The N1 of biotin is thought to be the active site base.
Site-directed mutagenesis
at D422 shows a change in the substrate specificity of the propionyl-CoA binding site, thus indicating this residue’s importance in PCC’s catalytic activity. In 1979, inhibition by phenylglyoxal
determined that a phosphate group from either propionyl-CoA or ATP
reacts with an essential arginine residue in the active site during catalysis. Later (2004), it was suggested that Arginine-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.
The KM values for ATP, propionyl-CoA, and bicarbonate has been determined to be 0.08 mM, 0.29 mM, and 3.0 mM respectively. The isoelectric point
falls at pH 5.5. PCC’s structural integrity is conserved over the temperature range of -50 to 37 degrees Celsius and the pH range of 6.2 to 8.8. Optimum pH was shown to be between 7.2 and 8.8 without biotin bound. With biotin, optimum pH is 8.0-8.5.
intermediate and does not proceed through a concerted process. Figure 3 shows a probable pathway.
The reaction has been shown to be slightly reversible at low propionyl-CoA flux.
s:
.
PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in assessment of biotin status.
(antiepileptic drug): significantly lowers enzyme levels in the liver
b. E. coli chaperonin
proteins groES and groEL: essential for folding and assembly of human PCC heteromeric subunits
c. Bicarbonate: negative cooperativity
d. Mg2+ and MgATP2-: allosteric activation
b. Glucokinase
in pancreatic beta cells: precursor of beta-PCC shown to decrease KM and increase Vmax; activation
Carboxylation
Carboxylation in chemistry is a chemical reaction in which a carboxylic acid group is introduced in a substrate. The opposite reaction is decarboxylation.-Carboxylation in organic chemistry:In organic chemistry many different protocols exist for carboxylation...
reaction of propionyl CoA in the mitochondrial matrix
Mitochondrial matrix
In the mitochondrion, the matrix contains soluble enzymes that catalyze the oxidation of pyruvate and other small organic molecules.The mitochondrial matrix also contains the mitochondria's DNA and ribosomes. The word "matrix" stems from the fact that this space is viscous, compared to the...
. The enzyme is biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
dependent. The product of the reaction is (S)-methylmalonyl CoA. Propionyl CoA is the end product of metabolism of odd-chain fatty acids, and is also a metabolite of most methyl-branched fatty acids. It is also the main metabolite of valine, and together with acetyl-CoA
Acetyl-CoA
Acetyl coenzyme A or acetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main function is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester...
, is a metabolite of isoleucine, as well as a methionine
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
metabolite. Propionyl-CoA
Propionyl-CoA
Propionyl-CoA is a coenzyme A derivative of propionic acid.-Production:There are several different ways in which it is formed:* It is formed as a product of beta-oxidation of odd-chain fatty acids....
is thus of great importance as a glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
precursor. (S)-Methylmalonyl-CoA is not directly utilizable by animals; it is acted on by a racemase to give (R)-methylmalonyl-CoA. The latter is converted by methylmalonyl-CoA mutase (one of a very few Vitamin B12 dependent enzymes) to give succinyl-CoA
Succinyl-CoA
Succinyl-Coenzyme A, abbreviated as Succinyl-CoA or SucCoA, is a combination of succinic acid and coenzyme A.-Source:It is an important intermediate in the citric acid cycle, where it is synthesized from α-Ketoglutarate by α-ketoglutarate dehydrogenase through decarboxylation...
. The latter is converted to oxaloacetate and then malate
Malate
Malate is the ionized form of malic acid. It is an important chemical compound in biochemistry. In the C4 carbon fixation process, malate is a source of CO2 in the Calvin cycle....
in the Krebs cycle. Export of malate
Malate
Malate is the ionized form of malic acid. It is an important chemical compound in biochemistry. In the C4 carbon fixation process, malate is a source of CO2 in the Calvin cycle....
into the cytosol leads to formation of oxaloacetate, phosphoenol pyruvate, and other gluconeogenic intermediates.
- ATP + propanoyl-CoA + HCO3- <=> ADP + phosphate + (S)-methylmalonyl-CoA
It has been classified both as a ligase
Ligase
In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...
and a lyase
Lyase
In biochemistry, a lyase is an enzyme that catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure...
.
Enzyme Structure
Propionyl-CoA Carboxylase (PCC) is a 750 kDa alpha(6)-beta(6)-dodecamer. (Only approximately 540 kDa is native enzyme. ) The alpha subunits are arranged as monomers, decorating the central beta-6 hexameric core. Said core is oriented as a short cylinder with a hole along its axis.The alpha subunit of PCC contains the biotin carboxylase
Biotin carboxylase
In enzymology, a biotin carboxylase is an enzyme that catalyzes the chemical reactionThe 3 substrates of this enzyme are ATP, biotin-carboxyl-carrier protein, and CO2, whereas its 3 products are ADP, phosphate, and carboxybiotin-carboxyl-carrier protein....
(BC) and biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
carboxyl carrier protein (BCCP) domains. A domain known as the BT domain is also located on the alpha subunit and is essential for interactions with the beta subunit. The 8-stranded anti-parallel beta barrel
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
fold of this domain is particularly interesting. The beta subunit contains the carboxyltransferase (CT) activity.
The BC and CT sites are approximately 55 Å apart, indicative of the entire BCCP domain translocating during catalysis of the carboxylation
Carboxylation
Carboxylation in chemistry is a chemical reaction in which a carboxylic acid group is introduced in a substrate. The opposite reaction is decarboxylation.-Carboxylation in organic chemistry:In organic chemistry many different protocols exist for carboxylation...
of propionyl-CoA
Propionyl-CoA
Propionyl-CoA is a coenzyme A derivative of propionic acid.-Production:There are several different ways in which it is formed:* It is formed as a product of beta-oxidation of odd-chain fatty acids....
. This provides clear evidence of crucial dimeric interaction between alpha and beta subunits.
The biotin-binding pocket of PCC is hydrophobic and highly conserved. Biotin and propionyl-CoA bind perpendicular to each other in the oxyanion hole containing active site. The native enzyme to biotin ratio has been determined to be one mole native enzyme to 4 moles biotin. The N1 of biotin is thought to be the active site base.
Site-directed mutagenesis
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
at D422 shows a change in the substrate specificity of the propionyl-CoA binding site, thus indicating this residue’s importance in PCC’s catalytic activity. In 1979, inhibition by phenylglyoxal
Phenylglyoxal
Phenylglyoxal is the organic compound with the formula C6H5CCH. It contains both an aldehyde and a ketone functional group. It is yellow liquid when anhydrous but readily forms a colorless crystalline hydrate...
determined that a phosphate group from either propionyl-CoA or ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
reacts with an essential arginine residue in the active site during catalysis. Later (2004), it was suggested that Arginine-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.
The KM values for ATP, propionyl-CoA, and bicarbonate has been determined to be 0.08 mM, 0.29 mM, and 3.0 mM respectively. The isoelectric point
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
falls at pH 5.5. PCC’s structural integrity is conserved over the temperature range of -50 to 37 degrees Celsius and the pH range of 6.2 to 8.8. Optimum pH was shown to be between 7.2 and 8.8 without biotin bound. With biotin, optimum pH is 8.0-8.5.
Enzyme Mechanism
The normal catalytic reaction mechanism involves a carbanionCarbanion
A carbanion is an anion in which carbon has an unshared pair of electrons and bears a negative charge usually with three substituents for a total of eight valence electrons. The carbanion exists in a trigonal pyramidal geometry. Formally a carbanion is the conjugate base of a carbon acid.where B...
intermediate and does not proceed through a concerted process. Figure 3 shows a probable pathway.
The reaction has been shown to be slightly reversible at low propionyl-CoA flux.
Isozymes
Humans express the following two propionyl-CoA carboxylase isozymeIsozyme
Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes...
s:
Pathology
A deficiency is associated with propionic acidemiaPropionic acidemia
Propionic acidemia, also known as propionic aciduria, propionyl-CoA carboxylase deficiency and ketotic glycinemia, is an autosomal recessive metabolic disorder, classified as a branched-chain organic acidemia....
.
PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in assessment of biotin status.
Of Propionyl-CoA Carboxylase
a. CarbamazepineCarbamazepine
Carbamazepine is an anticonvulsant and mood-stabilizing drug used primarily in the treatment of epilepsy and bipolar disorder, as well as trigeminal neuralgia...
(antiepileptic drug): significantly lowers enzyme levels in the liver
b. E. coli chaperonin
Chaperonin
Chaperonins are proteins that fold and unfold other proteins. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones...
proteins groES and groEL: essential for folding and assembly of human PCC heteromeric subunits
c. Bicarbonate: negative cooperativity
d. Mg2+ and MgATP2-: allosteric activation
By Propionyl-CoA Carboxylase
a. 6-Deoxyerythronolide B: decrease in PCC levels lead to increased productionb. Glucokinase
Glucokinase
Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver, pancreas, gut, and brain of humans and most other vertebrates...
in pancreatic beta cells: precursor of beta-PCC shown to decrease KM and increase Vmax; activation