Vitamins, Cosubstrates, and Coenzymes - Marquette University

Nucleotides, Vitamins, Cosubstrates, and Coenzymes

Objectives: I. Define the terms cofactor, activator ion, metalloenzymes, coenzyme, cosubstrate, prosthetic group,

vitamin, apoenzyme, and holoenzyme. II. Describe the structure; precursor vitamin, if present; biological function; and deficiency state for the

cosubstrates / coenzymes: A. Adenosine triphosphate (ATP) B. Guanosine triphosphate (GTP) C. Uridine Triphosphate (UTP) D. Cytidine Triphosphate (CTP) E. Nicotinamide adenine dinucleotide (NAD+) F. Nicotinamide adenine dinucleotide phosphate (NADP+) G. Flavin mononucleotide (FMN) H. Flavin adenine dinucleotide (FAD) I. Coenzyme A (CoA) J. Pyridoxal phosphate K. Thiamine pyrophosphate L. Tetrahydrofolate M. Biotin N. Lipoic acid O. Ascorbic acid P. Vitamin B12

Background - Review

Some enzymes require nonprotein components to attain full enzymatic activity. These nonprotein prosthetic groups can be metal ions or small organic molecules. Necessary metal ions are called Cofactors or Essential Ions. Cofactors tightly bound to the protein form Metalloenzymes. Those that are loosely associated with the protein are termed Activator Ions. The small organic molecules that are tightly bound or covalently linked to the protein are called Coenzymes. Cosubstrates are diffusible, they diffuse to and bind to the

Cofactors(Essential Ions)

Activator Ions(loosely bound)

Metaloenzymes(tightly bound)

Coenzymes(tightly bound)

(covalently linked)

Cosubstrates(loosely bound)

ProstheticGroups

Metal Ions Small Organics

©Kevin R. Siebenlist, 20181

protein during the catalytic cycle and then diffuse away to be used by other enzymes.

The APOENZYME is the protein part of the enzyme devoid of its required cofactor, cosubstrate, or coenzyme. The HOLOENZYME is the active functional enzyme, the protein and its necessary cofactor, cosubstrate, or coenzyme.

Coenzymes and Cosubstrates are often the metabolically active form of the vitamins. The word VITAMIN comes from VITAL AMINE. The first vitamins isolated were amines. Vitamins are small organic molecules that must be obtained from an outside source. Animals obtain vitamins from their diet and/or from the bacteria that colonize their gastrointestinal tracts.

There are numerous cosubstrates and coenzymes necessary for the proper functioning of cellular enzymes. A brief description of the most commonly encountered cosubstrates and coenzymes follows:

The Important Cosubstrates and Coenzymes

Nucleotides & Nucleosides General Considerations

When the term NUCLEOTIDE is mentioned in a biochemistry class, most students immediately think of either the precursors for the nucleic acids (ATP, GTP, CTP, UTP, dTTP) or the main energy carrying molecule of the cell (ATP). All Nucleotides are composed of / release upon hydrolysis:

• A Heterocyclic amine. The nitrogens of the molecule can have basic properties. • An aldopentose sugar - Ribose or 2-Deoxyribose. • One or more phosphate(s).

The heterocyclic base is linked to C-1 of the ribose or deoxyribose by an N-glycosidic bond (more on glycoside bonds when carbohydrates are examined). The first phosphate is linked to the hydroxyl group of C-5 of the sugar by a phosphoester bond. High energy phosphoanhydride bonds link the second phosphate to the first and the third to the second, if a second and/or third is present.

NUCLEOSIDES are formed when a heterocyclic base is covalently linked to the sugar. Nucleosides are converted to NUCLEOTIDES by the addition of a phosphate in phosphoester linkage to the 5´-hydroxyl group of the sugar.

Nucleotide triphosphates and Deoxynucleotide triphosphates are the precursors for RNA and DNA, respectively. Nucleotides are also:

• Energy storage and energy transfer molecules • Cosubstrates in transferase reactions • Carrier molecules of activated intermediates • Mediators of hormone action • Local hormones

ADENOSINE-5´-TRIPHOSPHATE. Adenosine-5´-Triphosphate (ATP) is considered by some the single most important molecule in the cell. Adenosine Triphosphate is a nucleotide composed of the heterocyclic base ADENINE covalently linked to carbon 1 of β-D-ribose (more about β-D-ribose in the up coming carbohydrates section). In ATP the first of three phosphate groups is linked to carbon five of ribose by a


low energy phosphoester bond. Two additional phosphate groups are covalently linked to the first phosphate by high energy phosphoanhydride bonds. The phosphate bonded to the ribose is the α phosphate, the middle phosphate is β, and the terminal one is the γ phosphate. Much of the chemical energy used by cells is stored in the two high energy phosphoanhydride bonds of ATP. ADENOSINE-5´-DIPHOSPHATE (ADP) is formed when the phosphoanhydride bond between the γ and β phosphate is hydrolyzed. ADENOSINE-5´-MONOPHOSPHATE (AMP) is formed when both high energy phosphoanhydride bonds are hydrolyzed. AMP is a low energy molecule.

ATP is the energy “currency” of the cell. Energy released by the hydrolysis of the high energy phosphoanhydride bonds in ATP drive many cellular reactions and cellular functions.

reactions that utilize ATP as a cosubstrate (second substrate) usually require Mg+2 as an activator ion. The Mg+2 ion salt bridges with the negative charges on the phosphates of ATP.

ATP can donate via transferase reactions phosphoryl (–PO3–2), pyrophosphoryl (–P2O6–3), adenylyl (AMP) or adenosyl groups (nucleoside).

N

NN

N

NH2

O

OHOH

CH2OP

O

OP

O

OP

O

O

O O O

N

NN

N

NH2

O

OHOH

CH2OP

O

OP

O

O

O O

N

NN

N

NH2

O

OHOH

CH2OP

O

O

O

N

NN

N

NH2

O

OH

CH2

O

O

P

O

O

Adenosine-5´-triphosphate

ATP

Adenosine-5´-diphosphate

ADP

Adenosine-5´-monophosphate

AMP

Adenosine-3´,5´-cyclicmonophosphate

Cyclic AMP

cAMP

N

NN

N

NH2

O

OHOH

CH2HO

N

NNH

N

O

CH3

OCH3

N

NN

N

O

CH3

OCH3

CH3

AdenosineCaffeine Theophylline


methionine is coupled to the adenosyl group to form S-Adenosylmethionine (SAM); SAM is an activated intermediate involved in many methylation (methyl group transferase) reactions. 3´,5´-cyclic AMP (cAMP), formed from ATP, is a second messenger produced intracellularly in response to a signal molecule binding to its receptor. Second messengers mediate a wide array of cellular responses by a variety of different mechanisms.

Finally, the nucleoside adenosine has signal molecule properties. It is a local hormone, an AUTOCOID, that when synthesized and released by a cell is bound to a receptor on nearby cells and causes:

1. blood vessel dilation 2. smooth muscle contraction 3. neurotransmitter release 4. increased fat metabolism

For example skeletal muscles undergoing rapid or prolonged muscular contraction releases adenosine causing the surrounding blood vessels to dilate and deliver more oxygen and nutrients to these tissues. When stressed cells produce and release adenosine. This molecule in the brain has a general overall calming effect on the organism and has been implicated in sleep regulation. During periods of prolonged wakefulness extracellular adenosine concentrations increase in the brain and this increase promotes sleepiness. CAFFEINE seems to function via the adenosine receptor. It binds to the receptor but does not stimulate a response or does not stimulate the complete response. CAFFEINE competes adenosine for binding to the receptor.

O

NH

N

N

O

NH2N

O

OH

CH2

OH

OP

O

O

POP

O O

O O

O

O

NH

N

N

O

NH2N

O

OH

CH2

OH

OP

O

O

PO

O

O

O

NH

N

N

O

NH2N

O

OH

CH2

OH

OP

O

O

NH

NN

N

O

OH

CH2

O

O

P

O

O

NH2

O

Guanosine-5´-triphosphate

GTP

Guanosine-5´-diphosphate

GDP

Guanosine-5´-monophosphate

GMP

Guanosine-3´,5´-cyclicmonophosphate

Cyclic GMP

cGMP


GUANOSINE-5´-TRIPHOSPHATE Guanosine-5´-Triphosphate (GTP) is a nucleotide containing the heterocyclic base GUANINE. Like ATP, GTP carries energy in phosphoanhydride bonds. In terms of cosubstrate/coenzyme activity, GTP is the “energy source” for the reactions of protein synthesis. GTP also serves an important role in the cellular response to signal molecules, e.g., hormones. 3´,5´-cyclic GMP (cGMP) formed from GTP is one of the many second messengers produced intracellularly in response to a signal molecule binding to its receptor. These second messengers mediate a wide array of cellular responses by a variety of mechanisms.

URIDINE-5´-TRIPHOSPHATE (UTP) Uridine-5´-Monophosphate (UTP) is a nucleotide with the heterocyclic base URACIL. UTP is used as a cosubstrate and activator/carrier molecule for monosaccharide isomerization (epimerization) reactions and for the biosynthesis of disaccharides and polysaccharides.

CYTIDINE-5´-TRIPHOSPHATE (CTP) Cytidine-5´-Monophosphate (CTP) is a nucleotide containing the heterocyclic base CYTOSINE. CTP is used as a cosubstrate and activator/carrier molecule during the biosynthesis of lipids (phospholipids and sphingolipids).

Nucleic acids are long linear polymers of nucleotides. When nucleic acids are hydrolyzed, nucleoside monophosphates result. The monomeric unit of nucleic acids are nucleoside monophosphates. Nucleoside triphosphates are the precursors for the synthesis nucleic acids. The energy released by the hydrolysis of the two phosphoanhydride bonds is used to form the 3´,5´-phosphodiester bond between the monomer units and to “drive” the reaction to completion.

DNA, Deoxyribonucleic acids, contain 2´-deoxyribonucleotides, whereas RNA, Ribonucleic acid, contain ribonucleotides. The backbone of the linear DNA and RNA polymers is comprised of repeating sugar and phosphate residues held together by 3´,5´ phosphodiester bonds. The heterocyclic bases on DNA are Adenine (A), Guanine (G), Cytosine (C), and Thymidine (T). In RNA thymidine is replaced by Uracil (U). Information of DNA and RNA is carried by the unique linear sequence of bases on the long linear polymer. At one end of each strand there is a 5´ triphosphate group that is not involved in a phosphodiester bond. This is the 5´ end of the molecule. At the other end there is a free, unreacted 3´ hydroxyl group. This is the

O

N

NH2

ON

O

OHOH

CH2OP

O

O

POPO

OO

O O

Cytidine-5´-triphosphate

CTP

O

NH

O

ON

O

OHOH

CH2OP

O

O

POPO

O O

O O

Uridine-5´-triphosphate

UTP


3´ end of the molecule

NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+) and NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADP+). These cosubstrates are composed of an AMP molecule covalently linked to a Nicotinamide Mononucleotide, hence dinucleotide. The nicotinamide part of the molecule comes from the vitamin NIACIN. NADP+ differs from NAD+ by the presence of an additional phosphate group on carbon two of the ribose ring of AMP. These molecules are diffusible cosubstrates that take part in oxidation / reduction reactions. NAD+ and NADP+ are the oxidized forms of the cosubstrate. When reduced, carbon four of the

DNA

RNA

O

NH

O

ON

O

OHO

CH2

PO

O

O

NH

N

N

O

NH2N

O

OH

CH2

O

PO

O

O

N

NH2

ON

O

OHO

CH2

P

O

O

N

NN

N

NH2

O

OHO

CH2

PO

O

O

N

NN

N

NH2

O

OHO

CH2

PO

O

O

N

NH2

ON

O

OHOH

CH2

O

POPOPO

O O O

OOO

O

NH

O

O

H3C

N

O

O

CH2

PO

O

O

NH

N

N

O

NH2N

OCH2

O

PO

O

O

N

NH2

ON

O

O

CH2

P

O

O

N

NN

N

NH2

O

O

CH2

PO

O

O

N

NN

N

NH2

O

O

CH2

PO

O

O

N

NH2

ON

O

OH

CH2

O

POPOPO

O O O

OOO


nicotinamide ring accepts a HYDRIDE (H–) ion, a proton and two electrons. NAD+/NADP+ always undergo two electron oxidations or reductions. NADH and NADPH are the abbreviation for the reduced forms.

The NAD+/NADH pair participates in oxidative reactions involved in energy production. NAD+ accepts electrons, is the oxidizing agent, during a particular metabolic reaction, the NADH formed during the first

Nicotinamide

(Vit - Niacin)

Ribose

AMP

Nicotinamide adenine dinucleotide

Oxidized (NAD+) Reduced (NADH)

Hydride Ion (H–)

Nicotinamide adenine dinucleotide phosphate

Oxidized (NADP+) Reduced (NADPH)

Hydride Ion (H–)


reaction is then used to reduce a substrate during a subsequent metabolic reaction. The NADP+/NADPH pair participates in reductive biosynthetic reactions. NADPH acts as the reducing agent.

If a vitamin is present at insufficient quantities or is completely lacking in the diet a deficiency disease often results. The effects of a diet lacking a single nutrient were determined using laboratory animals. The results were then extrapolated to the human animal. Whether a deficiency disease due to the lack of a single nutrient is possible in the human animal other than induced by a laboratory formulated diet is unclear. With that said, when Niacin is lacking the disease is called Pellagra. This condition is characterized by dermatitis on exposed areas; stomatitis; atrophic, sore, magenta tongue; impaired digestion; diarrhea; and disturbances of the CNS.

FLAVIN MONONUCLEOTIDE (FMN) and FLAVIN ADENINE DINUCLEOTIDE (FAD) {below} are both derived from the vitamin RIBOFLAVIN (B2). FMN is riboflavin linked to a modified form of the sugar ribose (the sugar alcohol ribitol) with a phosphate group esterified to the hydroxyl group of carbon five. FAD is FMN linked to an AMP molecule. FMN and FAD take part in oxidation / reduction reactions. The riboflavin ring usually undergoes a two electron oxidation or reduction. The flavin ring when reduced accepts a pair of hydrogen atoms, one at N-5 and the second at N-1 of the flavin ring. The reduced form of these molecules are FMNH2 or FADH2. This coenzyme can, under certain conditions, undergo one electron oxidations or reductions. For example, it can accept two electrons (2 H atoms) from a substrate in the first step of the reaction and then pass these two electrons (H atoms), one at a time to two different electron acceptor molecules. These molecules are usually covalently linked to their apoenzyme, hence they are considered coenzymes.

Flavin Ring

(Vit - Riboflavin)

Ribitol

Flavin mononucleotide

[oxidixed form] (FMN)

Flavin mononucleotide

[reduced form] (FMNH2)

2 Hydrogen Atoms


Riboflavin is not known to be the prime factor in a human deficiency disease. Riboflavin deficiency is characterized by a magenta tongue, fissuring at the corners of the mouth, seborrheic dermatitis, and corneal vascularization; symptoms similar to Pellagra. It is unclear whether this deficiency disease is due to a lack of riboflavin or due to a lack of several water soluble vitamins including niacin.

COENZYME A (abbreviated CoA or CoA-SH) is composed of an ADP molecule, linked to a molecule of PANTOTHENIC ACID (Vit B3) that is then linked to a molecule of 2-mercaptoethylamine. During the

Flavin Ring

(Vit - Riboflavin)

Ribitol

Flavin adenine dinucleotide

[oxidixed form] (FAD)

Flavin adenine dinucleotide

[reduced form] (FADH2)

2 Hydrogen Atoms

AMP


biosynthesis of CoA, the amino acid cysteine is amide linked to pantothenic acid and subsequently decarboxylated to form the 2-mercaptoethylamine. The active end of CoA is the free thiol (–SH) group of the 2-mercaptoethylamine. CoA carries carboxylic acids in thioester linkage. Since thioesters are high energy chemical bonds, the carboxylic acids carried by CoA are “activated” to undergo a variety of metabolic reactions. This molecule is a diffusible cosubstrate. No known deficiency states.

PYRIDOXAL PHOSPHATE is the biochemically active form of the vitamin PYRIDOXAL (Vit B6). Pyridoxal phosphate is the prosthetic group for a large number of enzymes that catalyze a wide variety of reactions involving amino acids. These reactions include isomerization, decarboxylation, side chain elimination, and/or amino group transfer (transamination). The formation of a Schiff base between the amino group on the substrate and the carbonyl group on pyridoxal phosphate is the first step in the reactions utilizing this coenzyme. The deficiency state is characterized by growth failure, dermatitis, edema, hypochromic anemia, convulsions, and CNS changes.

C

O

OHR C

O

OHR

ADP

Pantothenic Acid

Vitamin B3

2-Mercaptoethylamine

(from Cysteine)

NH

H2C

C

OH

CH3

O H

OPO

O

O

NH

H2C

C

OH

CH3

OPO

O

O

N

H

C

H

R CO

O

Pyridoxal phosphate

Schiff base between

Pyridoxal and

an amino acid


THIAMINE PYROPHOSPHATE (TPP) is the metabolically active form of the vitamin THIAMINE (B1). Carbon two of the thiazolium ring reacts with and carries molecules containing a ketone functional group. It is a necessary prosthetic group for enzymes that catalyze decarboxylation reactions of 2-ketocarboxylic acids (α-ketocarboxylic acids). It is also a prosthetic group for enzymes that transfer ketone groups between molecules (Transketolase reactions). The deficiency disease is BERIBERI. It is characterized by weight loss, muscle weakness, muscle wasting, and peripheral neuritis. Sensory changes, anxiety states, and mental confusion can occur.

TETRAHYDROFOLATE is the biologically active form of the vitamin FOLIC ACID (FOLATE). The folate molecule is composed of a conjugated PTERIN RING system coupled to 4-AMINOBENZOIC ACID which is linked to from one to six GLUTAMATE molecules. Tetrahydrofolate (TH4) is important in one carbon metabolism. It accepts and donates a variety of one carbon fragments bonded to N-5, N-10, or both. These one carbon fragments are donated to a variety of acceptor molecules. The one carbon fragments carried by TH4 are at the oxidation levels of methyl, methylene, methanol, formaldehyde, or formic acid. The deficiency state is characterized by growth failure, anemia, leukopenia, or pancytopenia.

BIOTIN was originally called vitamin H. Biotin serves as a coenzyme for enzymes that catalyze carboxyl-group-transfer reactions and ATP dependent carboxylation reactions (the addition of CO2). The cofactor is usually covalently linked to the enzyme by an amide bond to the amino group on a lysine side chain. The usually substrate for the carboxylation reaction is the bicarbonate ion (HCO3–). Mechanistically, the

Thiamine pyrophosphate

N

N

H2C N

SNH2

H3CH3C

CH2

H2C O P O P O

O O

O O

H

Tetrahydrofolate

Pterin Ring

System

Glutamate

Aminobenzoic

Acid

C

H2C

CH2

CH

CO O

NH

C

O

NH

H2C

HN

O

HN

H2N N NH

O

O

N5

N10


reaction involves at least two steps In the first step bicarbonate ion is activated by reaction with ATP and covalent linkage to N-1 of the biotin ring system. The lysine side chain tether then moves the activated biotin-carboxyl complex to a different region of the active site where the carboxyl group is passed to an acceptor molecule.

LIPOIC ACID can be synthesized in adequate amounts by mammals, so by the strictest definition it is not a vitamin. Lipoic acid is 6, 8-DITHIOOCTANOIC ACID. The thiols can exist in a five membered oxidized disulfide ring system or in a reduced (open) form. When active it is covalently linked to the enzyme by an amide bond to the side chain of a lysine residue. Lipoic acid is involved in oxidation / reduction reactions and it carries carboxylic acids to and from active sites in multi-enzyme complexes.

VITAMIN C or ASCORBIC ACID has been discussed in the context of collagen biosynthesis. It is a necessary coenzyme for the hydroxylation of proline (Prolyl Hydroxylase) and the hydroxylation of lysine (Lyslyl Hydroxylase) during collagen biosynthesis. Vitamin C also functions as an anti-oxidant within the cell.

The deficiency state is SCURVY. The symptoms of scurvy in adults include spongy gums, loosening of the teeth, bleeding into the skin and mucous membranes due to increased capillary fragility, and decreased immunocompetence. Bone development in children is abnormal resulting in bowed legs and/or stunted growth. With a severe or prolonged deficiency of vitamin C there is decreased wound healing, osteoporosis, hemorrhaging, and anemia.

VITAMIN B12 or COBALAMINE takes part in only two reactions in humans. It is necessary for the Methionine

S

NHC

HN

O

CH2

H2C

CH2

H2C

CO

OBiotin

S S

H2C

CH2

H2C

CH2

C

O

O

H2C

CH2

H2C

CH2

C

O

O

SH SHLipoic Acid (oxidized form) Dihydrolipoic Acid (reduced form)

Vitamin C or Ascorbic Acid


Synthase a transferase reaction which methylates homocysteine to methionine and it is necessary for L-Methylmalonyl-CoA Mutase which converts L-methylmalonyl-CoA to succinyl-CoA. Vitamin B12 deficiency results in the accumulation of both homocysteine and L-methylmalonyl-CoA. B12 deficiency presents with megaloblastic anemia with neurological deterioration caused by progressive demyelination. Vitamin B12 is widespread in foods of animal origin, especially meats. Since the liver can store up to a six year supply, deficiencies are rare except in older individuals and in long term vegetarians.

N

NH

N

N

Co3+

CH3CH2

CH2

C

H3C

H3C

H2C

H2CC

H2N

O

CH3CH2

C

H3CCH2

CH2

CH2N O

CH3

CH2

C NH2

O

H2C C

NH2

O

CH2

CH3

CH2

C O

NH

CH2

HC CH3

O

P O

N

N

H2C

OHO

OH

H3C

H3C O

OH

NH2

O

N

N N

N

NH2

O

OH OH

H3C

H2N

O


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Vitamins, Cosubstrates, and Coenzymes - Marquette University