enzymology An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

, formate C-acetyltransferase (''pyruvate formate lyase'') () is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

. Pyruvate formate lyase is found in ''

Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...

'' and other

organism An organism is any life, living thing that functions as an individual. Such a definition raises more problems than it solves, not least because the concept of an individual is also difficult. Many criteria, few of them widely accepted, have be ...

s. It helps regulate anaerobic

glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...

metabolism Metabolism (, from ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cellular processes; the co ...

. Using radical non-redox chemistry, it catalyzes the reversible conversion of

pyruvate Pyruvic acid (CH3COCOOH) is the simplest of the alpha-keto acids, with a carboxylic acid and a ketone functional group. Pyruvate, the conjugate base, CH3COCOO−, is an intermediate in several metabolic pathways throughout the cell. Pyruvic ...

and coenzyme-A into

formate Formate (IUPAC name: methanoate) is the conjugate base of formic acid. Formate is an anion () or its derivatives such as ester of formic acid. The salts and esters are generally colorless. Fundamentals When dissolved in water, formic acid co ...

and

acetyl-CoA Acetyl-CoA (acetyl coenzyme A) is a molecule that participates in many biochemical reactions in protein, carbohydrate and lipid metabolism. Its main function is to deliver the acetyl group to the citric acid cycle (Krebs cycle) to be oxidation, o ...

. The reaction occurs as follows: This enzyme belongs to the family of

transferase In biochemistry, a transferase is any one of a class of enzymes that catalyse the transfer of specific functional groups (e.g. a methyl or glycosyl group) from one molecule (called the donor) to another (called the acceptor). They are involved ...

s, specifically those acyltransferases transferring groups other than aminoacyl groups. The

systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...

of this enzyme class is acetyl-CoA:formate C-acetyltransferase. Other names in common use include pyruvate formate-lyase, pyruvic formate-lyase, and formate acetyltransferase. This enzyme participates in 3

metabolic pathways In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell. The reactants, products, and intermediates of an enzymatic reaction are known as metabolites, which are modified by a sequence of chemical ...

: pyruvate metabolism, propanoate metabolism, and butanoate metabolism.

Structural studies

As of late 2007, 8 structures have been solved for this class of enzymes, with PDB accession codes , , , , , , , and . Pyruvate formate lyase is a

homodimer In biochemistry, a protein dimer is a macromolecular complex or protein multimer, multimer formed by two protein monomers, or single proteins, which are usually Non-covalent interaction, non-covalently bound. Many macromolecules, such as proteins ...

made of 85 kDa, 759-residue subunits. It has a 10-stranded beta/alpha barrel motif into which is inserted a beta finger that contains major catalytic residues. The

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

of the enzyme, elucidated by

x-ray crystallography X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

, holds three essential amino acids that perform catalysis ( Gly734, Cys418, and Cys419), three major residues that hold the substrate pyruvate close by ( Arg435, Arg176, and Ala272), and two flanking hydrophobic residues ( Trp333 and Phe432).Becker A., Fritz-Wolf K., Kabsch W., Knappe J., Schultz S., Volker wagner A.F. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. 1999 Nat. Struct. Biol. 6: 969–975. Studies have found structural similarities between the active site of pyruvate formate lyase and that of Class I and Class III

ribonucleotide reductase Ribonucleotide reductase (RNR), also known as ribonucleoside diphosphate reductase, is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. It catalyzes this formation by removing the 2'-hydroxyl group of the ribos ...

(RNR) enzymes.

Mechanism

Roles of the three catalytic residues

It has been shown that:Becker, A., Kabsch W. X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA. How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage. 2002 J Biol Chem. 277(42): 40036–42.Plaga, W., Wielhaber, G., Wallach, J., Knappe, J. Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism. 2000 FEBS Lett. 466(1): 45–8. * Gly734 (glycyl radical) – transfers the radical on and off Cys418, via Cys419 * Cys418 (thiyl radical) – does acylation chemistry on the carbon atom of the pyruvate

carbonyl In organic chemistry, a carbonyl group is a functional group with the formula , composed of a carbon atom double bond, double-bonded to an oxygen atom, and it is divalent at the C atom. It is common to several classes of organic compounds (such a ...

* Cys419 (thiyl radical) – performs hydrogen-atom transfers

Steps

# The proposed mechanism for pyruvate formate lyase begins with radical transfer from Gly734 to Cys418, via Cys419. # The Cys418 thiyl radical adds covalently to C2 (second carbon atom) of pyruvate, generating an acetyl-enzyme intermediate (which now contains the radical). # The acetyl-enzyme intermediate releases a formyl radical that undergoes hydrogen-atom transfer with Cys419. This generates formate and a Cys419 radical. # coenzyme-A comes in and undergoes hydrogen-atom transfer with the Cys419 radical to generate a coenzyme-A radical. # The coenzyme-A radical then picks up the acetyl group from Cys418 to generate acetyl-CoA, leaving behind a Cys418 radical. # Pyruvate formate lyase can then undergo radical transfer to put the radical back onto Gly734. Note that each step is reversible.

Regulation

Two additional enzymes regulate the “on” and “off” states of pyruvate formate lyase to regulate anaerobic glucose metabolism: pyruvate formate lyase activase (AE) and pyruvate formate lyase deactivase. Activated pyruvate formate lyase allows formation of acetyl-CoA, a small molecule important in the production of energy, when pyruvate is available. Deactivated pyruvate formate lyase, even with substrates present, does not catalyze the reaction. Pyruvate formate lyase activase is part of the radical SAM ( S-adenosylmethionine) superfamily. The enzyme turns pyruvate formate lyase “on” by converting Gly734 (G-H) into a Gly734 radical (G^*) via a 5'- deoxyadenosyl radical (via a

radical SAM Radical SAM enzymes belong to a superfamily of enzymes that use an iron-sulfur cluster (4Fe-4S) to reductively cleave S-Adenosyl methionine, ''S''-adenosyl-L-methionine (SAM) to generate a radical (chemistry), radical, usually a 5′-deoxyadenosyl ...

). For more information about radical SAM activation and

enzymes, see the discussion by Wang et al., 2007. Pyruvate formate lyase deactivase turns pyruvate formate lyase “off” by quenching the Gly734 radical. Furthermore, pyruvate formate lyase is sensitive to molecular

oxygen Oxygen is a chemical element; it has chemical symbol, symbol O and atomic number 8. It is a member of the chalcogen group (periodic table), group in the periodic table, a highly reactivity (chemistry), reactive nonmetal (chemistry), non ...

(O₂), the presence of which shuts the enzyme off.Zhang, W., Wong, KK., Magliozzo, RS., Kozarich, JW. Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR. 2001 Biochemistry 40(13): 4123–30.

References

{{Portal bar, Biology, border=no EC 2.3.1 Enzymes of known structure