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Propionyl Coenzyme-A Carboxylase

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 (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 A 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 showed 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.

Crystallography

Enzyme Mechanism
The normal catalytic reaction pathway involves a carbanion intermediate and does not proceed through a concerted process. The diagram shown below is accurate with two minor exceptions: (1) the first step shown should be split in two, with the phosphorylation of bicarbonate happening before the biotinyl-enzyme attacks, and (2) the resonance structure given for propionyl-CoA is not the major resonance structure.

Mechanism Diagram

The reaction has been shown to be slightly reversible at low propionyl-CoA flux.

Pathology

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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.

Graphs from study

Of Propionyl-CoA Carboxylase
a. Carbamazepine (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

By Propionyl-CoA Carboxylase
a. 6-Deoxyerythronolide B: decrease in PCC levels lead to increased production b. Glucokinase in pancreatic beta cells: precursor of beta-PCC shown to decrease KM and increase Vmax; activation