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Subject Chemistry
Paper No and Title Paper 16, Bioorganic and biophysical chemistry
Module No and Title Module 26, Co-enzyme II
Module Tag CHE_P16_M26
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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TABLE OF CONTENTS
1. Learning outcomes 2. Introduction 3. Nicotinamide nucleotides 3.1 Mechanism of oxido-reduction by NAD+/NADP+ 3.1 Examples of NAD+/NADP+ dependent enzymes 4. Co-enzyme FMN and FAD 3.2 Metabolic role as Co-enzyme 3.3 FMN dependent Co-enzyme 5. Lipoic acid 6. Summary
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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1. Learning Outcomes
After studying this module, you shall be able to
Know the three important Co-enzymes i.e. Nicotinamide nucleotides, Flavin nucleotides and Lipoic acid. Know the mechanism of oxido-reduction by NAD+/NADP+. Understand NADH and NADPH dependent reaction. Learn about the metabolic role of Co-enzyme.
2. Introduction
This group consist of three important coenzymes all of which assist different enzymes in oxidation- reduction reactions.
These are
a. Nicotinamide nucleotides
b. Flavin nucleotides
c. Lipoic acid
The dehydrogenation of an alcohol to ketone or aldehyde is one of the most frequent biological oxidation- reduction reactions.
Although the hydrogen atoms removed from substrate are often indicated simply as 2[H], it was recognized early in the twentieth century that they are actually transferred to hydrogen carrying coenzymes such as NAD+, NADP+,FAD+, FMN, lipoic acid etc. They may be considered as carriers of hydrogen or as carriers of electrons [H = H+ + e-] in metabolic reactions.
When NAD+ becomes reduced by dehydrogenation of alcohol, one of the hydrogen atoms removed from alcohol becomes firmly attached to the NAD+, converting it to NADH, the other is released as proton. CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Why are there four major hydrogen transfer coenzymes, NAD+, NADP+, FAD and riboflavin phosphate [FMN], instead of just one? Part of the answer is that reduced pyridine nucleotides NADPH and NADH are more powerful reducing agents than are reduced flavins conversely, flavin coenzymes are more powerful oxidizing agents than are NAD+ and NADP+. This difference reflects the chemical difference between the vitamins riboflavin and nicotinamide which form the oxidation - reduction centres of coenzymes. Another difference is that NAD+ and NADP+ tend to be present in free forms within cells, diffusing from a site of one enzyme to a site on another. These coenzymes are sometimes tightly bound but flavin coenzymes are usually firmly bound to proteins, fixed and unable to move. Thus, they tend to accepts hydrogen atom from one substrate and to pass them to a second substrate while attached to a single enzyme.
3. Nicotinamide nucleotides
These coenzymes are derived from vitamin, niacin [vitamin B3]. Niacin is synonymous with nicotinic acid
Fig.1 Nicotinic acid
NAD+ and NADP+ are the active form of niacin. They are called nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate respectively. Collectively, they are called pyridine nucleotides
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Fig.2 Structure of NAD+ and NADP+
NAD+ is a dinucleotide made up to two nucleotides joined by phosphodiester bond. One nucleotide is made up of nicotinamide [N-base] with ribose and phosphate. The second is AMP.
In NADP+ the second carbon-hydroxyl group of ribose of AMP is phosphorylated to form NADP -Nicotinamide, nicotinic acid and tryptophan are all the precursors of synthesis of nicotinamide mononucleotide (NMN) which is then converted to NAD+ and NADP+. In the oxidized form the N-atom of nicotinamide is positively charged and written as NAD+ and NADP+. In the reduced form this charge is neutralized. So they are in form of NADH and NADPH.
3.1 Mechanism of oxido-reduction by NAD+ / NADP+
Two hydrogen atom are transferred from the substrate of NAD+ or NADP+. One H-atom is transferred as hydride ion (H-) to pyridine ring to nicotinamide whose positive charge in neutralized. The other proton goes free in medium as a byproduct. So only one hydrogen atom is attached to pyridine ring. That is why reduced NAD+ is always written as NADH + H+ but not as NADH2.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Why are these two pyridine nucleotide, NAD+ or NADP+, differing only in presence or absence of extra phosphate group? One important answer is that they are members of two different oxidation reduction systems, both based on nicotinamide but functionally independent. The experimentally measured ratio of [NAD+]/[NADH] is much higher than the ratio [NADP+] / [NADPH]. Thus, these two coenzymes systems can operate within a cell at different redox potentials. A related generalization that holds much of time is that NAD+ is usually involved in pathways of catabolism, where is functions as an oxidant, while NADPH often used as reducing agent in biosynthetic processes. Typically NADPH is said to participate in reductive biosynthesis.
3.2 Examples of NAD+ / NADP+ dependent enzymes
Table1. Examples of NAD+ / NADP+ dependent enzymes
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Some examples of NAD+ / NADH and NADP+ / NADPH + H+ dependent reactions
1.
2.
3.2.1 NADH dependent reaction
NADPH dependent reactions are usually reductions in biosynthetic reaction. Hence these are called as reductive biosynthetic reactions.
3.2.2 NADPH-Dependent Reactions
B-keto acyl - CoA dehydrogenase in fatty acid biosynthesis. HMG-CoA reductase in cholesterol synthesis. CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Phenylalanine hydroxylase for conversion of phenylalanine to tyrosine. Dihydrofolate reductase for synthesis of tetrahydrofolate from folic acid.
4. Coenzyme FMN and FAD
Flavin nucleotides are derived from vitamin B2 [riboflavin] and are actively involved in hydrogen transfer reactions.
Fig.4 Structure of FMN and FAD
Riboflavin is 6, 7 dimethyl-iso-alloxan bound to ribitol [alcohol of ribose]. The intense yellow color is due to iso-alloxan ring.
Active co-enzyme forms are FMN and FAD
FMN [flavin mono-nucleotide] and FAD [flavin adenine dinucleotide] are the active or coenzyme form of riboflavin.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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FMN is formed by phosphorylation of riboflavin by kinase using ATP in intestine
4.1 Metabolic role as Co-enzyme
Riboflavin containing proteins are called flavoproteins. Many oxido-reductase enzymes are flavoproteins containing FMN and FAD as prosthetic group. Example L-amino oxidase [FMN dependent], succinate dehydrogenase in citric acid cycle is FAD+ dependent. These enzymes participate in reversible oxidation reduction reactions. N-atoms at position 1 and 10 in isoalloxan ring of riboflavin undergo oxidation reduction
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Example of enzymes requiring FMN or FAD as a coenzyme and reaction where they are involved.
Table.2 role of different flavoprotien enzymes in different reactions
4.2 FMN dependent enzymes
4.2.1 L- amino acid oxidases
They catalyze oxidative deamination of amino acid producing H2O2 which is split by catalase.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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4.2.2 NADH dehydrogenase complex
This enzyme is a part of electron transport chain and contains FMN. The electrons are transferred from NADH to FMN and then to CoQ in the electron transport chain
Reactions of FAD dependent enzymes:
1.
2.
3.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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4.
5. Lipoic acid
Lipoic acid acts in the transfer of hydrogen during oxidative decarboxylation reactions.
Fig.6 Lipoic acid
It occurs both in oxidized and reduced forms as shown in fig.7.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Fig.7 Oxidized and reduced form of lipoic acid
Also fig.8 shows the structure of lipoamide, where lipoic acid is bound in the amide linkage to the ε amino group of lysine residue of dehydrolipoamide acyl transferases. The complex reactions of the carbohydrate metabolism catalyzed by pyruvate dehydrogenase system and l-ketoglutarate dehydrogenase require participation of lipoic acid. It acts as a carrier and undergoes inter conversion between reduced and oxidized form as shown in fig.7.
Fig.8 Lipoamide
The two thiol groups that can undergo reversible oxidation to a disulfide (-s-s-), similar to that between two Cyc residues in a protein. Because of its capacity to undergo oxidation- reduction reactions, lipoate can serve both as an electron hydrogen carrier and as an acyl carrier.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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Fig.9 Acetylated form
Lipoic acid is also known as α-Lipoic acid or ALA and thioctic acid. Lipoic acid is cofactor for atleast five enzymes systems, two of these are in the citric acid cycle through which many organisms turn nutrients into energy e.g.
The pyruvate dehydrogenase complex The α-ketoglutarate dehydrogenase The branched chain oxoacid dehydrogenase (BCDN) complex. (4) acetoin dehydrogenase complex.
The most studied of these is pyruvate dehydrogenase complex
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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These complex have three central subunits (E1) pyruvate dehydrogenase, E2 (Dihydrolipoyl transacetylase (E3) dihydrolipoyl dehydrogenase (E3). These complex have a central E2 core and other subunits surround this core to form complex. In the gap between these two subunits, the lipoyl domain ferries intermediates between active site. The lipoyl domain itself is attached to a flexible linker to E-2 core.
6. Summary
Three important coenzymes which assist in oxidation-reduction are Nicotinamide nucleotides, flavin nucleotides and lipoic acid. Riboflavin is a component of FMN and FAD, whereas niacin is present in NAD and NADP. Lipoic acid is also known as ALA (α-lipoic acid) or thioctic acid. NAD+ is a dinucleotide made of two nucleotides joined by phosphodiester bond, one nucleotide is made up of nicotinamide (N-base) with ribose and phosphate and second is AMP. NADP+ the second carbon-hydroxyl group of ribose of AMP is phosphorylated to form NADP+ Two hydrogen atom are transferred from substrate to NAD+ or NADP+ one H-atom is transferred as hydride ion (:H-) to pyridine ring to nicotinamide, the other proton goes
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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free in medium as H+ and reduced form of NAD+ & NADP+ is written as NADH + H+ and NADPH + H+ NAD+ is usually used in pathway of catabolism as oxidant NADPH often used as reducing agent in biosynthetic processes. FAD and FMN has iso-alloxan ring which accepts H2 from substrates and converted to FADH2 and FMNH2 NAD+, FAD+, FMN, NADP+ and Lipoic acid serves as coenzymes for oxidoreductase enzymes involved in carbohydrate, protein, lipid, nucleic acid metabolism and electron transport chain.
CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II
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CHEMISTRY PAPER No. 16 Bio-organic and biophysical chemistry MODULE No.26 Coenzyme- II