Enzyme cofactors and co-substrates

• Amino acid side chains (R groups) are the site of reactivity for enzymes, there are 20 of them.

• However the 20 R groups are not enough for all the different types of chemical reactions required by enzymes.

• Cofactors increase the number and type of functional reaction centres in enzymes compared to the 20 naturally occurring side chains of AAs.

• Apoenzyme + cofactor à holoenzyme.

• Apoenzyme(inactive) + cofactor = holoenzyme (active)

•Some apoenzymes require both metal ions and coenzymes.

• Amino acids that are implicated directly in enzymatic catalysis are those with functional groups on the side-chains.

• Protein functional groups include

• hydroxyl, serine [I], threonine [II], tyrosine [III]

• sulfhydryl, cysteine [IV]

• ammonium, lysine [V] and arginine [VI]

• imidazolium, histidine [VII]

• carboxylate, aspartate [VIII] and glutamate [IX]

• amide, asparagine [X] and glutamine [XI] and

• thio-ether, methionine [XII]. 

• Nearly half of all enzyme reactions are known to depend on at least one cofactor,

• cofactors are clearly crucial to the ability of enzymes to perform catalytic reactions.

• To define what a cofactor constitutes the International Union of Pure and Applied Chemistry (IUPAC) Gold Book states that cofactors are

• “organic molecules … or ions … that are required by an enzyme [for] its activity.”

• This definition also states that a

• “cofactor binds with its associated protein (apoenzyme), which is functionally inactive, to form the active enzyme (holoenzyme)”.

cofactors are split in two groups essentail ions and coenzymes 

essentail ions include activator ions and metal ions of metalloenzymes (tightly bound) 

coenzymes include sosubstrates (loosely bound) and prosythetic groups (tihgtly bound) 

• A molecule that can;

• transfers electrons,

• is dissociable,

• is not a standard amino acid

• is actively involved in catalysis.

• The Gold Book defines a coenzyme as “the dissociable,

Low relative molecular mass active group of an enzyme which

transfers chemical groups, hydrogen or electrons.”

• In contrast, a prosthetic group is “the non-amino acid

portion of a conjugated protein

• Only those prosthetic groups that are located in the

active site of an enzyme are denoted cofactors.

• A prosthetic group “may be attached either

loosely or tightly… to the enzyme.”

• For our purpose, a prosthetic group is therefore distinguished from a coenzyme in that, once inserted into the apoenzyme, a prosthetic group remains with the enzyme.

• In contrast, a coenzyme binds to the enzyme at the

beginning of the catalytic cycle and then leaves the active

site after each cycle.

cofactors are split into two types - inorganic complexes e.g metal cofactors and organi ccomplexes 

organic complexes are split into Aii - prosthetic group stays with enzyme and Ai - coenzyme dissociable 

Aii is split into Aiii polypeptide derived and non-amino acid based 

• The catalytic activity of the majority of enzymes is attributed to the activity of AA residues forming active site domain.

• BUT other factorsaffect this catalytic activity, these factors often participate in catalysis and are called cofactors.

• They convert inactive apoenzymesto active holoenzymes.

• There are two types of cofactors.

• Essential ions, mostly metal ions, mainly divalent cations.

• Coenzymes, organic compounds.

• Both the above form essential parts of the active site domain of the enzymes.

• Some enzymes require both a coenzyme and a metal ion to be active

• Both can be either loosely or tightly bound to the enzyme.

• Loosely bound cofactors function as

  co-substrates.

• While tightly, even covalently, bound ones are known asprosthetic groups.

• Remember that:

–An active enzyme together with its bound coenzyme and/or metal ion is called a holoenzyme. –While the protein part only of this enzyme is called an apoenzyme.

• Mostly metalions.

• Many minerals required by organisms are essential, some are called activator ions.

• Activator ions are reversibly bound and are important for the substrate binding.

• Some cations are tightly bound and take part in the catalytic process.

• More than 25% of enzymes require a metal ion  for their activity.

• They can be metal-activated enzymes, they have an absolute requirement for or are stimulated by a metal ion: K+, Ca2+, Mg2+

• Or they can bemetalloenzymesthat have ions tightly bound to their active site: Fe2+, Zn2+ 

• Metal ions areessentialfor the catalytic reaction.

• They can act as electrophilic catalysts.

Or they can undergo oxidation-reduction reactions.

• Participate in the catalytic process by:

1. binding to substrate to orient them properly for the reaction.

2. mediating oxidation-reduction reactions through reversible changes in the metal ions oxidation state.

3. electrostatically stabilizing or shielding negative charges,

4. often metal ion acts similar to a proton, or polarizes water to generate OH

• Coenzymes are specific group-transfer agents.

• The groups can be H or e-or they can transfer a far larger covalently linked group.

• The group is usually attached to the reactive center of the coenzyme.

• They are classifiedaccording to their interaction with the apoenzyme.

• Two ways to classify coenzymes.

• Classification due to interactionbetween the apoenzyme and coenzyme – 2 types

–Co-substrates. –Prosthetic groups.

• And according to the source of the coenzyme.

–Metabolite. –Vitamin Derived.

• Act as substrates in the enzyme catalysed reaction.

• They are altered and dissociatefrom the active site during reactions.

• They are regenerated by a further reaction.

• They are recycled repeatedly in the cell.

• They shuttlemetabolic groups.

• They remain tightly boundto the enzyme during the reaction.

• They can be covalently linked or tightly bound by many weak interactions.

They have to return to their original formin order for the holoenzyme to retain its activity

• Synthesized from common metabolites.

• Include nucleoside triphosphateses,

–ATP

• S-adenosylmethionine

• Nucleotide sugars,

–UDP-glucose.

• Vitamins are nutritional substances that an animal cannot synthesis.

• Vitamins are required for the synthesis of coenzymes.

• The reaction sometimes involves ATP, the part of ATP transferred to the coenzyme is the group that binds it to the enzymes active site.

• The nicotinamidecoenzymes.
• NAD+, nicotinamide adenine dinucleotide.

• NADP+, nicotinamide adenine dinucleotide phosphate.

• The first coenzymes discovered.

• Derived from niacin, nicotinic acid.

• They are usually the coenzymes for dehydrogenases / redox reactions.

•The riboflavincoenzymes - vitamin B2. •FAD, flavin adenine dinucleotide. •FMN, flavinmononucleotide. •Are required by many enzymes catalysingoxidation-reduction reactions.

They can donate electrons, either one or two at a time 

• Historically known as the acetylation coenzyme, hence the name coenzyme A.

• Requires B vitamin pantothenic acid

•Required for many metabolic processes: –Oxidation of metabolic fuels. –Biosynthesis of some carbohydrates & lipids. •Involved in acyl-group transferreactions. •Contains an –SH group which is the active center. •Forms part of an important protein prosthetic group, phosphor-pantotheine.

• Thiamine pyrophosphate, TPP.

• Derived from thiamin, B1.

• Required by a number of enzymes:

–Decarboxylases / carboxylases. –Oxidative decarboxylation of a-keto acids. –Prosthetic group fortransketolases. –It forms the prosthetic group of yeastpyruvate decarboxylase.

• In animals it is synthesised by transfer of pyrophosphoryl group from ATP to thiamine.

• The prosthetic group of enzymes catalysing:

–Carboxyl-group transfer reactions. –ATP-dependent carboxylation reactions.

• Covalently bound to an amino group of a lysine residue.

• It works as a CO2 carrier.

• It is synthesised by intestinal bacteria, therefore very rare deficiency in humans.

• Coenzymes of the vitamin folate, they are the reduced form and they contain additional glutamate residues.

• Glutamate residues help bind the coenzyme to the enzyme and keeps the molecule inside the cells.

• Formed from folate by adding hydrogen to positions 5,6,7 & 8 by reduction in two NADH dependent steps catalysed by dihydrofolate reductase.

• Required by enzymes catalysing the transfer of one C-unit in oxidation of:

–Methanol, CH3OH. –Formaldehyde, HCHO. –Formic acid, HCOOH.

• Another pterin coenzyme is 5,6,7,8-tetra-hydrobiopterin,

a cofactor for many hydroxylases and a reducing agent in the conversion of phenylalanine to tyrosine

• Cobalamin, vitamin B12.

• Corrin ring system similar to porphyrin ring of heme.

• Required by all animals as a micronutrient.

• Participates in many enzyme reaction catalysingintramolecular re-arrangment.

• An exchange of a hydrogen atom and a group attached to an adjacent C takes place.

• Lipoamide is the protein bound form of lipoic acid, vitamin B !.

• Does not occur free but is found covalently attached to a lysine residue in a protein.

• Forms part of the multi-enzyme complexes of the citric acid cycle, pyruvate and a-ketoglutarate dehydrogenase complexes.

• Acts as a swinging arm carrying acyl groups between the active sites of the enzyme complexes.

• Coenzyme Q.

• Is a benzoquinone with a long isoprenoid side chain.

• Synthesised by respiring organisms and photosynthetic bacteria.

• Occurs in lipid bilayers and is found in the inner mitochondrial membrane, it transports e-s between enzyme complexes.

• It can acceptordonate one or two electrons at a time
• When accepting 1 electron it becomes a semi-quinone radical (.QH).
• When accepting 2 electrons it becomes ubiquinol (QH2).
• It then transfers these accepted electrons to the other components of the ETC.

• Certain proteins, cytochromes, thioredoxins, carboxyl-carrier proteins etc. are coenzymes.

• They do not catalyse reactions but are required by enzymes.

• They are called group-transfer proteins or protein coenzymes.

• They participate in oxidation reduction reactions were the transferred group is H or e-.

• The reactive centers are metal ions, iron-sulfur clustersandheme groups.

• They can also contain parts of or other coenzymes,biotinand lipoamide.

• Protein coenzymes, the active center is a hemegroup.

• They participate in electron transfer reactions.

• This is measured as a reduction potential that can be identified from their absorption spectra.