The following points highlight the five main enzymes involved in oxidative process. The enzymes are: 1. Oxidases 2. Aerobic Dehydrogenases 3. Anaerobic Dehydrogenases 4. Hydro-peroxidases 5. Oxygenases.
Oxidative Processes: Enzyme # 1. Oxidases:
(a) Enzymes that catalyse the removal of hydrogen from a substrate but use only oxygen as a hydrogen acceptor to form water as a reaction product (with the exception of uricase and monoamine oxidase which form H2O2).
(b) They are conjugated proteins containing copper as prosthetic groups.
(i) Cytochrome oxidase:
(a) Cytochrome oxidase is a hemoprotein widely distributed in plants and animal tissues.
(b) It is the terminal component of respiratory chain found in mitochondria.
(c) It is poisoned by cyanide and hydrogen sulfide.
(d) More recent studies show that 2 cytochromes are combined with the same protein and the complex is known as cytochrome aa3.
(e) Cytochrome aa3 contains 2 molecules of heme A, each having one Fe atom. 2 atoms of Cu are also present which are associated with the cytochrome oxidase activity.
(f) The terminal cytochrome aa3 is responsible for the final combination of reducing equivalents with molecular oxygen.
(g) This enzyme system contains copper, a component of several oxidase enzymes.
(h) It has a high affinity for oxygen.
(i) It is the only one in the chain which signifies the irreversible reaction.
(j) It gives direction to the movement of reducing equivalents in the respiratory chain and to the production of ATP, to which it is coupled.
(ii) Phenolase (tyrosinase, polyphenol oxidase, cotechol oxidase):
(a) It is a copper-containing enzyme.
(b) It converts monophenol to O-quinones.
(a) It is widely distributed in plants and animals.
(b) It converts P-hydroquinone’s to P-quinones.
(c) It also contains copper.
(iv) Ascorbic oxidase:
(a) It contains copper.
(b) It is found only in plants.
(a) It also contains copper.
(b) It catalyzes the oxidation of uric acid to allantoin.
(vii) Monoamine oxidase:
(a) It is found in the mitochondria of several tissues.
(b) It oxidizes epinephrine and tyramine.
Oxidative Processes: Enzyme # 2. Aerobic Dehydrogenases:
(a) They catalyse the removal of hydrogen from a substrate and use either oxygen or artificial substances such a methylene blue as hydrogen acceptor.
(b) H2O2 is formed as a product.
(c) They are flavoprotein enzymes having FMN (flavin mononucleotide) or FAD (Flavin adenine dinucleotide) as prosthetic groups.
(d) Many of the flavoprotein enzymes contain a metal for which they are known as metalloflavoprotein enzymes.
(i) D-amino acid dehydrogenase (D-amino acid oxidase):
(a) It is an FAD-linked enzyme.
(b) It is found particularly in liver and kidney.
(c) It catalyzes the oxidative deamination of the unnatural (D-) forms of amino acids.
(ii) L-amino acid dehydrogenase (L-amino acid oxidase):
(a) It is an FMN-linked enzyme.
(b) It is found in kidney.
(c) It catalyzes the oxidative deamination of naturally occurring L-amino acids.
(iii) Xanthine dehydrogenase (Xanthine oxidase):
(a) It occurs in milk and liver.
(b) In the liver, it converts purine bases to uric acid.
(c) It contains FAD as the prosthetic group.
(d) It is highly significant in the liver and kidneys of birds which excrete uric acid as the end product of purine metabolism and also of protein and amino acid catabolism.
(e) It is a metalloflavoprotein containing non-heme iron and molybdenum.
(f) It also oxidizes all aldehydes.
(iv) Aldehyde dehydrogenase (aldehyde oxidase):
(a) It is an FAD-linked enzyme.
(b) It is present in pig and other mammalian liver.
(c) It is also a metalloflavoprotein containing nonheme iron and molybdenum.
(d) It oxidizes aldehydes.
(v) Glucose oxidase:
(a) It is an FAD-linked enzyme.
(b) It is prepared from fungi.
(c) It is used in estimating glucose.
Oxidative Processes: Enzyme # 3. Anaerobic Dehydrogenases:
(a) They catalyze the removal of hydrogen from a substrate but not able to use oxygen as hydrogen acceptor.
(b) They transfer hydrogen from one substrate to another by oxidation-reduction reaction not involving a respiratory chain (shown in Fig. 12.4.).
(c) They perform oxidation of metabolite utilizing several components of a respiratory chain (shown in Fig. 12.5).
(i) Dehydrogenase dependent on Nicotinamide Coenzymes:
(a) They are linked as coenzymes either to NAD (Nicotinamide adenine dinucleotide) or to NADP (Nicotinamide adenine dinucleotide phosphate).
(b) The coenzymes are reduced by the particular substrate of the dehydrogenase and re-oxidized by a suitable electron acceptor and synthesized from the vitamin niacin (nicotinic acid and nicotinamide).
(c) NAD-linked dehydrogenases catalyze oxidoreduction reactions in glycolysis, the citric acid cycle and in the respiratory chain of mitochondria.
(d) NADP-linked dehydrogenases are found in fatty acid and steroid synthesis in the extra-mitochondria. They are also found in hexose monophosphate shunt.
(e) Some nicotinamide coenzyme-dependent dehydrogenases contain zinc, particularly alcohol dehydrogenase from liver and glyceraldehyde-3-phosphate dehydrogenase from skeletal muscle. The zinc ions do not take part in the oxidation and reduction.
(ii) Dehydrogenases dependent on Riboflavin Prosthetic Groups:
(a) Most of the riboflavin-linked anaerobic dehydrogenases are concerned with electron transport in the respiratory chain.
(b) Succinate dehydrogenase, acyl-CoA dehydrogenase and mitochondrial glycerol- β-phosphate dehydrogenase transfer electrons directly from the substrate to the respiratory chain.
(c) In the dehydrogenation of reduced lipoate, an intermediate in the oxidative decarboxylation of pyruvate and α-Ketogluta-rate, the flavoprotein (FAD) due to the low redox potential acts as a carrier of electrons from reduced lipoate of NAD. The electron transferring flavoprotein in an intermediary carrier of electrons between acyl-CoA dehydrogenase and the respiratory chain.
(iii) The Cytochromes:
(a) The cytochromes excepting cytochrome oxidase are anaerobic dehydrogenases. They are involved as carriers of electrons from flavoproteins to cytochrome oxidase in the respiratory chain.
(b) They are iron-containing hemoproteins in which iron becomes Fe+++ and Fe++ oxidation and reduction. The cytochromes in the respiratory chain are b, c1, c, a and a3.
(c) Cytochromes are also found in the endoplasmic reticulum (cytochromes P-450 and b5) plant cells, bacteria and yeast.
1. It has a molecular wt. of 13,000.
2. The iron porphyrin group of cytochrome c is attached to protein more firmly than in the hemoglobin.
3. It is quite stable to heat and acids.
4. The reduced form of cytochrome c is not auto-oxidizable.
5. The peptide chain of human heart cytochrome c contains 104 amino acids. Acetyl glycine is the N-terminal amino acid and glutamic acid is the c-terminal amino acid. Two cysteine residues are located at positions 14 and 17. The linkage of iron in heme occurs through the imidazole nitrogen of histidine residue at position 18 in the peptide chain.
1. Hepatic ALA synthase is markedly increased on the administration of many drugs to humans. Most of these drugs are metabolized in the liver by a system that utilizes a specific hemoprotein, Cytochrome P450. The utilization of heme by cytochrome P450 is highly increased during the metabolism of these drugs and in turn diminishes the intracellular heme concentration.
2. Hydroxylation reactions take place by the enzymes mono-oxygenases or cytochrome P450.
The reaction catalyzed by a mono-oxygenase (cytochrome P450) is:
[RH represents a very wide variety of xenobiotics including drugs, carcinogens, pesticides, petroleum products, and pollutants].
3. Cytochrome P450 is considered the most versatile biocatalyst. It has been shown by the use of 1802 that one atom of oxygen enters R-OH and one atom enters water. This dual fate of the oxygen is responsible for naming of mono-oxygenases as “mixed-function oxidases”.
The reaction catalyzed by cytochrome P450 is represented:
4. The major mono-oxygenases in the endoplasmic reticulum are cytochrome P-450s—so named because the enzyme was prepared from microsomes that had been chemically reduced and exhibited a distinct peak at 450 nm on exposure to carbon monoxide. About 50 per cent of the drugs that humans ingest are metabolized by isoforms of cytochrome P450; these enzymes also act on various carcinogens and pollutants.
5. About 150 isoforms of cytochrome P450 have been discovered so far. The abbreviated root symbol CYP denotes a cytochrome P450. CYPA1 denotes a cytochrome P450 that is a member of family 1 and subfamily A and is the first individual member of that subfamily.
6. Like hemoglobin, they are hemoproteins.
7. These are widely distributed among species. Bacteria possess cytochrome P450s, and P450cam (involved in the metabolism of camphor) of pseudomonas putida is the only P450 isoform.
8. They are present in large amount in liver. They are present mainly in the membranes of the smooth endoplasmic reticulum in liver and most other tissues. In the adrenal, they are found in mitochondria as well as in the endoplasmic reticulum; the various hydroxylases present in that organ play an important role in cholesterol and steroid biosynthesis.
The mitochondrial cytochrome P450 system differs form the microsomal system in that it uses an NADPH-linked flavoprotein, adrenodoxin reductase, and a non-heme iron-sulphur protein, adrenodoxin.
9. There are at least six isoforms of cytochrome P450 present in the endoplasmic reticulum of human liver, each is acting on both xenobiotics and endogenous compounds. In recent years, the genes for many isoforms of P450 have been isolated and studied.
10. NADPH is involved in the reaction mechanism of cytochrome P450. The enzyme that uses NADPH to form the reduced cytochrome P450 is called NADPH-cytochrotne P450 reductase. Electrons are transferred from NADPH to NADPH-cytochrome P450 reductase and then to cytochrome P450. This leads to the reductive activation of molecular oxygen, and one atom of oxygen is subsequently inserted into the substrate.
11. Lipids are also components of the cytochrome P450 system. The suitable lipid is phosphatidyl choline which is found in membranes of the endoplasmic reticulum.
12. Most isoforms of cytochrome P450 are inducible. The mechanism of induction involves increased transcription of mRNA for cytochrome P450. Induction of cytochrome P450 has important clinical implications, since it is a biochemical mechanism of drug interaction.
The example of enzyme induction is CYP2E1 which is induced by consumption of ethanol. This P450 metabolizes certain widely used solvents and also components found in tobacco smoke, many of which are established carcinogens. If the activity of CYP2E1 is elevated by induction, this may increase the risk of carcinogenicity.
13. Certain isoforms of cytochrome P450 (e.g., CYP1 Al) are particularly involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs) and related molecules. That is why they were formerly called aromatic hydrocarbon hydroxylases (AHH).
These enzymes are important in the metabolism of (PAHs) and in carcinogenesis produced by these agents. Smokers have higher levels of this enzyme in some of their cells and tissues than do non-smokers. Some reports have mentioned that the activity of this enzyme may be elevated (induced) in the placenta of a woman who smokes, thus altering the quantities of metabolites of PAHs to which the fetus is exposed.
14. Recently, it has been shown that certain cytochrome P450s exist in polymorphic forms, some of which exhibit low catalytic activity. These observations are the important explanation for the variations in drug responses noted among many patients.
One P450 exhibiting polymorphism is CYP2D6 which is involved in the metabolism of debrisoquin (an antihypertensive drug). Certain polymorphisms of CYP2D6 cause poor metabolism of these and many other drugs, so that they can accumulate in the body resulting in awful consequences.
Oxidative Processes: Enzyme # 4. Hydro-peroxidases:
They utilize hydrogen peroxide as a substrate.
Two enzymes fall into this category:
(a) It is found in milk and leukocytes and the prosthetic group is protoheme.
(b) It catalyzes the reduction of hydrogen peroxide by the help of ascorbic acid, quinones and cytochrome C which act as electron acceptors.
The reaction is complex but the overall reaction is:
(a) It is hemoprotein and found in blood and liver.
(b) It uses one molecule of H2O2 as a substrate electron donor and another molecule as electron acceptor.
(c) Its function is to destroy H2O2 formed by the action of aerobic dehydrogenases.
Oxidative Processes: Enzyme # 5. Oxygenases:
They catalyze the incorporation of oxygen into a substrate molecule.
(i) Dioxygenases (Oxygen transferases, true oxygenases)
(a) They catalyze the incorporation of two atoms of oxygen (O2) into the substrate:
(b) Enzymes containing iron as a prosthetic group e.g., homogentisate di-oxygenase, 3-hydro-xyxanthranilate di-oxygenase and enzymes utilizing heme as a prosthetic group such as L-tryptophan di-oxygenase (tryptophan pyrrolase) from the liver.
(ii) Mono-oxygenase (Mixed function oxidases, Hydroxylases):
(a) They catalyze the incorporation of only one atom of the oxygen molecule into a substrate. The other oxygen atom is reduced to water.
A cosubstrate is necessary for this purpose:
(b) Many of the enzymes involved in steroid synthesis are mono-oxygenase using NADPH as a cosubstrate. They are found mainly in the endoplasmic reticulum (Microsomes) of the liver and in mitochondria and the microsomes of the adrenal glands.
(c) They are also involved in the metabolism of many drugs by hydroxylation. They are found in the microsomes of the liver together with cytochrome P450 and cytochrome b5. The drugs metabolized by this system are benzpyrine, aminopyrine, aniline, morphine and benzphetamine.
But phenobarbital induce the formation of microsomal enzymes and of cytochrome P450:
(d) They are concerned with the synthesis or degradation of many different types of metabolites.