Coenzymes

• Many enzymes contain small nonprotein molecules and metal ions

• Coenzyme – a complex organic or metalloorganic molecule

• Prosthetic groups– are distinguished by their tight, stable incorporation into

a protein’s structure by covalent or noncovalent forces.– Examples

• Pyridoxal phosphate,flavin mononucleotide (FMN), flavin dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions.

• Some enzymes require both a coenzyme and one or more metal ions for activity

• Coenzymes– participate directly in substrate binding or

catalysis.

• A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme. The protein part of such an enzyme is called the apoenzyme or apoprotein.

• Intracellular range of concentration– 0.01 -1.0 meq L-1

• Coenzymes may be covalently bound to the enzyme or free to associate and dissociate from the protein.

• may facilitate the binding and orientation of substrates

• the formation of covalent bonds with reaction intermediates (Co2+ in coenzyme B12)

• interaction with substrates to render them more electrophilic (electron-poor) or nucleophilic (electron-rich).

• Coenzymes serve as recyclable shuttles—or group transfer reagents—that transport many substrates from their point of generation to their point of utilization.– Other chemical moieties transported by coenzymes include

methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides (dolichol).

• Association with the coenzyme also stabilizes substrates such as hydrogen atoms or hydride ions that are unstable in the aqueous environment of the cell.

• Chemical reactions of many types take place between substrates and enzymes’ functional groups (specific amino acid side chains, metal ions, and coenzymes).

• Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and activate it for reaction, or a group may be transiently transferred from the substrate to the enzyme.

• A number of amino acid side chains, and the functional groups of some enzyme cofactors can serve as nucleophiles in the formation of covalent bonds with substrates.

• The covalent bond formed can activate a substrate for further reaction

Coenzyme role

• Epimerization involves an oxidation and reduction at carbon 4 with NAD+ as coenzyme.

• Each step in fatty acid oxidation involves acyl-CoA derivatives catalyzed by separate enzymes, utilizes NAD+ and FAD as coenzymes, and generates ATP

• Susceptibility to proteolytic degradation can be influenced by the presence of ligands such as substrates, coenzymes, or metal ions that alter protein conformation

• NAD is also required for the poly-ADP-ribose polymerase reaction, which is part of the cellular DNA damage recognition system and regulates DNA replication, DNA repair, and cell cycle progression.

• Metabolic pathways are regulated at several levels, from within the cell and from outside– The most immediate regulation is by the

availability of substrate – A second type of rapid control from within is

allosteric regulation by a metabolic intermediate or coenzyme—an amino acid or ATP, for example—that signals the cell’s internal metabolic state.

• Many coenzymes, cofactors, & prosthetic groups are derivatives of B vitamins

• Many coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP.

Many coenzymes and related compounds are derivatives of adenosine monophosphate.

Synthesis

• They are synthesized by a variery of ammalian cell types

• Synthesis of nucleotide coenzymes is regulated so that there are essentially constant concentrations of these coenzymes in the cell.

• Synthesis of niacin requires pyridoxine, riboflavin, and iron.

• The function of coenzymes is chemically varied, and we describe each separately

• The coenzyme pyridoxal phosphate (PLP) is present at the catalytic site of aminotransferases and of many other enzymes that act on amino acids

• In each case, the thiamin diphosphate provides a reactive carbon on the thiazole moiety that forms a carbanion, which then adds to the carbonyl group of, for instance, pyruvate. The addition compound then decarboxylates, eliminating CO2.

• Electrical stimulation of nerve leads to a fall in membrane thiamin triphosphate and release of free thiamin. It is likely that thiamin triphosphate acts as a phosphate donor for phosphorylation of the nerve membrane sodium transport channel.

• NAD Is the Source of ADP-Ribose– ADP-ribosylation – DNA repair mechanism

• Vitamin B6 Has Several Roles in Metabolism– transamination and decarboxylation– glycogen phosphorylase, where the phosphate group

is catalytically important – in steroid hormone action

• where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones.

• A few types of coenzymes and proteins serve as universal electron carriers

• Both NAD and NADP accept two electrons and one proton.

• the coenzymes function catalytically and are recycled repeatedly without a net change in the concentration of NAD NADH.

• NAD and NADP are the freely diffusible coenzymes of many dehydrogenases.

• NAD or NADP accepts a hydride ion (:H-, the equivalent of a proton and two electrons)

• the coenzymes function catalytically and are recycled repeatedly without a net change in the concentration of NAD+ + NADH.

• NAD and NADP are bound to dehydrogenases in a widely conserved structural motif called the Rossmann fold.

• Coenzymes play key roles In the citric acid cycle– energy-yielding metabolism

• NAD(P)+-dependent dehydrogenases are assayed spectrophotometrically– the rate of change in optical density at 340 nm will

be proportionate to the quantity of enzyme present

• Many enzymes that do not directly reduce NAD+ or FAD generate products that can be acted upon by a NAD(P) or FAD-linked dehydrogenase. Thus by coupling two enzyme reactions

Sirtuins

• The sirtuins are a highly conserved family of NAD+-dependent enzymes

• sirtuins serve as the bridge between what we eat and what we are.

• Sirtuin activity is intimately tied to the metabolic state of the cell.– Linking Chromatin Remodeling to Metabolism

• requires NAD+ cleavage with each reaction cycle

• flavoproteins as electron carriers• Certain flavoproteins act in a quite different

role as light receptors.– Mediate the effects of light on mammalian

circadian rhythms (oscillations in physiology and biochemistry, with a 24-hour period)• Photolyases use the energy of absorbed light to repair

chemical defects in DNA.

• Vitamin C Is the Coenzyme for Two Groups of Hydroxylases– Copper-containing hydroxylases – α-ketoglutarate-linked iron-containing

hydroxylases

CLINICAL CORRELATION

• Cystathioninuria• Mutation of a coenzyme-binding site results in

clinical disease – y-cystathionase

• Cystathionine → cysteine + α-ketobutyrate• the Km for pyridoxal phosphate binding to the

enzyme was increased

Arsenic Poisoning

• For the most part, arsenic poisoning is explained by inhibition of enzymes that require lipoic acid as a coenzyme. These include pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain a-keto acid dehydrogenase.

Deficiency of vitamines

• limited diets, when food is cooked at high temperatures for long periods,

• intestinal diseases, Inability to absorb• in newborns , pregnant women• Folic Acid Deficiency– Altered appearance of blood cells and

formiminoglutamate excretion

• methyl malonic aciduria– Acidosis– 5’-adenosylcobalamin deficiency (coenzyme of

methyl malonyl CoA isomerization).

• methylmalonic aciduria

Assay of vitamines(coenzyme)

• by measuring one or more enzyme activities in isolated red blood cells

As a tool for enzyme Purification

• Stationary phase matrices available commercially contain ligands such as NAD+ or ATP analogs.

• Bound proteins are then eluted either by competition with soluble ligand or, less selectively, by disrupting protein-ligand interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentrations.