5 Biotransformationby enzymes

Dr. Yogalakshmi K N and Dr. K N Sangeetha

Objectives:

To familiarize the concept of biotransformation
To study the process of enzymatic biotransformation
To familiarize the properties, distribution and stereochemical aspects of enzymes involved in biotransformation

Biotransformation

Xenobiotic is defined as a chemical or molecule that is foreign to and exerts a variety of effects on the biological system. These effects may be beneficial, in the case of drugs, or deleterious, in the case of poisons. The origin of these toxins that are present in our bodies commonly referred to as xenobiotics comes from an array of sources. These sources include exogenous sources that include environmental exposure; such as in the air we breathe, the food we eat, the water we drink, and medications; and the endogenous sources such as; the products produced by digestion, energy metabolism, tissue regeneration, and end products from the metabolism of hormones, bacterial by-products and other complex molecules. Other xenobiotics includes natural and manufactured chemicals such as drugs, industrial chemicals, pesticides, pollutants, pyrolysis products in cooked food, alkaloids, secondary plant metabolites, and toxins produced by molds, plants, and animals. Xenobiotics are mostly lipohilic in nature due to which they are easily absorbed through the skin, lungs, or gastrointestinal tract and cannot be easily eliminated from the body. Detoxification is the process of transforming and removing xenobiotics that are potentially harmful for the body. Elimination of xenobiotics often depends on their conversion to water-soluble chemicals. So the process of transforming toxins into a form suitable for excretion is called biotransformation. Biotransformations are structural modifications of a chemical compound by organisms /enzyme systems that lead to the formation of molecules with relatively greater polarity. The chemical process through which the endogenous or exogenous chemicals are modified into water soluble products, so that they can be easily eliminated from the body. Without biotransformation, lipophilic xenobiotics would be excreted from the body so slowly that they would eventually overwhelm and kill an organism.

Natural Biotransformation mechanisms are catalyzed by enzymes in the liver and other tissues. However, the natural transformation process is slow, nonspecific and less productive. The most significant aspect of biotransformation is that it maintains the original carbon skeleton after obtaining the products.

Enzyme induction in biotransformation

Xenobiotic biotransformation is the principal mechanism for maintaining homeostasis during exposure of organisms to small foreign molecules, such as drugs. In general, xenobiotic biotransformation is accomplished by a limited number of enzymes with broad substrate specificities. The synthesis of some of these enzymes is triggered by the xenobiotic by the process of enzyme induction. However, in most of the cases the enzymes are expressed constitutively (i.e., they are synthesized in the absence of a discernible external stimulus). Enzyme induction is an adaptive and reversible response to xenobiotic exposure. It enables some xenobiotics to accelerate their own biotransformation and elimination. The drugs which bring about these changes are known as enzyme inducers. Some examples include anticonvulsants like phenytoin, carbomycin and chronic alcoholism. Others include various sedatives, hypnotics, tranquilizers and insecticides. Likewise, certain drugs exhibit auto-metabolism, a process in which certain drugs are taken together where one acts as enzyme inducers for other causing increased metabolism of them. For example, Artemether and carbamazepine when taken together exhibits auto-metabolism.

Enzyme induction is brought about by gene transcription. Enzyme induction leads to decreased levels of the parent drug and increased levels of metabolites. At certain times, the process also results in the formation of toxic metabolites. The rate of reaction increases thereby converting the parent drug faster. Generally, drugs following first pass metabolism, have decreased bioavailability.

Some enzyme inducers include:

Ethchlorvynol enhances metabolites of warfarin
Phenytoin enhances metabolism of cortisol and digitoxin
Rifampicin increases metabolism of digitoxin
Barbiturates
Chloral hydrate
Erythromycin

Consequences of Enzyme Induction

The consequences of enzyme induction include

Decreased intensity and duration for action of drugs e.g. failure of contraceptives
Increased intensity of action of drugs activated by metabolism. E.g. acute paracetamol toxicity is due to one of its metabolites.
If drug induces its own metabolism e.g. cicobarbitone it develops tolerance so effects are not produced.
Precipitation of acute intermittent porphyria. Enzyme induction might increase porphyrin synthesis.
Intermittent use of an inducer might interfere adjustment of dose of another drug e.g. oral anti coagulants, oral hypoglycemic, antiepileptics and antihypertensives.

Auto induction

Auto induction is a phenomenon in which a drug induces metabolism of other drugs and its own. E.g. carbamazepine-antiepileptic.

Enzyme Inhibition

It is defined as a process in which metabolizing capacity of an enzyme is decreased thereby decreasing the rate of metabolism of the drug. Drugs that bring about these changes are known as enzyme inhibitors. Examples include ketoconazole – antifungal drug, cimetidine and verapamil – calcium channel blocker. Enzyme inhibition is a rapid process and is considered most critical for the drugs having a large therapeutic index. Competitive binding of the drug (enzyme inhibitor) occurs at the active site of the enzyme receptor and thereby leads to decreased metabolism. The decrease in the metabolic rate of the drug increases the levels of parent drug and decreases the levels of metabolites. At certain times, serious drug-drug interactions might occur due to increased half-life of plasma.

Example:

Sulfonamides decrease the metabolism of phenytoin so that its blood levels become toxic
Cimetidine decreases the metabolism of propanolol leading to enhanced bradycardia.
Metabolism of tolbutamide is inhibited by phenylbutazone or coumarins, leading to prolonged hypoglycemia
Oral contraceptives inhibit metabolism of antipyrine

Properties of xenobiotic biotransforming enzymes

Xenobiotic biotransforming enzymes are limited with broad substrate specificities. The enzymes are usually expressed constitutively but sometimes the synthesis of some of these enzymes is triggered by the xenobiotic itself by the process of enzyme induction. The specificity of xenobiotic biotransforming enzymes is so broad that they metabolize a large variety of endogenous chemicals, such as ethanol, acetone, steroid hormones, vitamins A and D, bilirubin, bile acids, fatty acids, and eicosanoids.

Xenobiotic biotransforming enzymes, or enzymes that are closely related, play an important role in the synthesis of many of these same molecules. For example, several steps in the synthesis of steroid hormones are catalyzed by cytochrome P450 enzymes in steroidogenic tissues. In general, these steroidogenic enzymes play little or no role in the biotransformation of xenobiotics. In the liver, however, other cytochrome P450 enzymes convert steroid hormones to water-soluble metabolites that are excreted in urine or bile, in an analogous manner to the biotransformation and elimination of xenobiotics.

The structure (i.e., amino acid sequence) of a given biotransforming enzyme may differ among individuals, which can give rise to differences in rates of xenobiotic biotransformation. In general, a variant form of a xenobiotic biotransforming enzyme (known as an allelic variant or an allelozyme) has diminished enzymatic activity when compared to wild-type enzyme. However, the impact of amino acid substitution on the catalytic activity of a xenobiotic biotransforming enzyme is usually substrate-dependent, such that an allelic variant may interact normally with some substrates (and inhibitors) but abnormally with others. The study of the causes, prevalence, and impact of heritable differences in xenobiotic biotransforming enzymes is known as pharmacogenetics.

Distribution of xenobiotic biotransforming enzymes

Xenobiotic biotransforming enzymes are widely distributed throughout the body. These enzymes are also located in the skin, lung, nasal mucosa, eye, and gastrointestinal tract. Their location is rationalized on the basis that these are major routes of exposure to xenobiotics. They are also expressed in several other tissues such as the kidney, adrenal, pancreas, spleen, heart, brain, testis, ovary, placenta, plasma, erythrocytes, platelets, lymphocytes and aorta. In vertebrates, the liver is the richest source of enzymes catalyzing biotransformation reactions. Within the liver, the enzymes catalyzing xenobiotic biotransformation reactions are located primarily in the endoplasmic reticulum or the cytoplasm, with lesser amounts in mitochondria, nuclei, and lysosomes. Their presence in the endoplasmic reticulum can be rationalized on the basis that the xenobiotics requiring biotransformation for urinary or biliary excretion will be lipophilic and, hence, soluble in the lipid bilayer of the endoplasmic reticulum. Intestinal microflora plays an important role in the biotransformation of certain xenobiotics. Likewise, nasal epithelium plays an important role in the biotransformation of inhaled xenobiotics, including odorants. It is quantitatively unimportant in the biotransformation of orally ingested xenobiotics.

Stereochemical Aspects of Xenobiotic Biotransformation

Xenobiotics especially drugs exits as enantiomers or stereoisomers as they contain one or more chiral centers. Biotransformation of chiral xenobiotics occurs stereoselectively i.e., one enantiomer is biotransformed faster than its antipode. For example, in humans the antiepileptic drug Mesantoin, a racemic mixture of R- and S-mephenytoin, is biotransformed stereoselectively, such that the S-enantiomer is rapidly hydroxylated and eliminated faster than the R-enantiomer. Likewise, xenobiotic biotransforming enzymes can also be inhibited stereoselectively by some chiral xenobiotics. For example, for quinidine drug, a potent inhibitor of a human cytochrome P450 enzyme its enantiomer quinine, has relatively little inhibitory effect.

In cases of achiral xenobiotics, they are first converted to a mixture of enantiomeric metabolites stereoselectively such that one enantiomer is formed preferentially over its antipode. Example, several cytochrome P450 enzymes catalyze the 6 hydroxylation of steroid hormones. Some P450 enzymes (such as CYP2A1) preferentially catalyze the 6α hydroxylation reaction, whereas other P450 enzymes (such as CYP3A) preferentially catalyze the 6β hydroxylation reaction which is considered the major route of hepatic steroid biotransformation.

Biotransformation reactions

During the biotransformation process, xenobiotics interacts with the biotransforming enzymes and may change the toxicant to either a less or a more toxic form. These reactions catalyzed by xenobiotic biotransforming enzymes are generally divided into two groups, called phase I and phase II. These phase I and II reactions may occur simultaneously or sequentially.

Phase I Reactions

Phase I reaction is a catabolic process that breaks down the toxicant into various components. It involves hydrolysis, reduction, and oxidation that introduce polar chemical moieties either by inserting new polar functional groups (–OH, –NH2, –SH or –COOH) or by interchanging or unmasking existing functional groups that results in a small increase in hydrophilicity. Phase I reactions are non-synthetic in nature and in general produce a more water-soluble and less active metabolites.

Oxidation

Hydroxylation

Hydroxylation of aromatic ring – e.g. phenobarbitone is converted into p-hydroxy phenobarbitone
Aliphatic hydroxylation – e.g. Meprobamate is converted into hydroxymeprobamate

Dealkylation

Conversion of mephobarbitone into phenobarbitone

O-Dealkylation

Conversion of codeine into morphine.

N-oxidation:

Conversion of aniline into nitrobenzene

Sulfoxidation

Conversion of chlorpromazine into chlorpromazine sulfoxide

Deamination

Conversion of amphetamine into phenylactate.

Desulfuration

Conversion of parathion into paraoxon

Reduction

Chloramphenicol, dantrolene, clonazepam

Hydrolysis

Esters: procaine, suxamethonium and aspirin

Amides: procainamide, lidocaine

Phase I reactions are mediated by enzymes such as cytochrome P-450, FMO, esterases and amidases. CYP enzymes are the most important enzymes responsible for the pharmacological activation of many drugs. Many toxicants are metabolized by the enzymes cytochrome P-450 reductase and cytochrome P-450 in association with NADPH, a co-enzyme present in most cells and interacts with various substances during normal cell metabolism. These two enzymes are found in abundance in the endoplasmic reticulum of liver cells. When these enzymes interact with the toxic molecule, one atom of oxygen is attached to the toxic molecule and another oxygen atom interacts with hydrogen to form water.

Cytochrome P450

Cytochrome P450 (CYP450) is the main element in the liver’s metabolic process. They are widely distributed among tissues especially located in the mitochondria and endoplasmic reticulum. They are present in all living things, including viruses, bacteria, animals, plants, and fungi. CYP450 comprises of family of proteins that exists in multiple forms. They have very broad specificity and can metabolize many xenobiotics. They are also responsible for catalysing many types of reactions. These enzymes are active in transforming multiple substrates that metabolize about 90% of the drugs that are consumed.

Their levels can be increased based on their exposure to chemicals in the food, water, or air. These enzymes are essential to the metabolism of vitamin D, hormones, cholesterol, and fatty acids. They function in the metabolism of prostacyclins and thromboxane, which are key to the process of blood clotting. CYPs are also critical in the body’s ability to detoxify foreign chemicals. CYPs are responsible for deactivating drugs in several ways by oxidizing the substrate, by catalyzing the substrate or by assisting in excreting substrate waste. CYPs can also change an initial drug or pro-drug into an active form that the body can use. Many nutrients, medications, and herbal therapies are metabolized through the CYP450 enzyme system.

Consequences of Phase I reactions:

Active drug may be converted into inactive metabolite. Active parent drug inactivation may terminate biological activity.

Active drug may be converted into active metabolite. E.g. morphine is converted into more active metabolite.

Prodrug may be converted into active metabolite

Active drug may be converted into toxic metabolite

e.g. halothane used in general anesthesia, is converted into trifluoroacetylated compound or trifluoroacetic acid, leading to hepatic toxicity.

Biotransformation of xenobiotics to mutagenic or carcinogenic agents. Conversion of xenobiotics into harmless compound.

For every 10 molecules of cytochrome P450, only one NADPH cytochrome reductase is present.

Phase II reactions

In phase II reactions, small endogenous polar molecules (e.g. glucuronic acid, glycine and sulfate) conjugate with the functional groups formed during phase I reactions to form less toxic water soluble

P450.Cytochrome P450 cycle, RH is parent drug, ROH is oxidized metabolite, e- is electron.

compounds thereby favouring excretion. This type of reaction is referred to as conjugation. Direct conjugation of endogenous molecules can also occur if the compound already contains appropriate functional groups. These conjugative reactions are mediated by enzymes such as glucuronosyltransferase, sulfotransferase and N-acetyltransferase. Most phase II biotransformation reactions result in a large increase in xenobiotic hydrophilicity, hence they greatly promote the excretion of foreign chemicals. Phase II biotransformation of xenobiotics may or may not be preceded by phase I biotransformation.

There are several types of Phase II reactions, of which glucuronidation is probably the most important. Conjugation with glucuronidation occurs as a result of the conjugation of glucuronidation acid with either a metabolite from Phase I reactions or with the parent compound. There are several chemical compounds that interact with glucuronic acid such as alcohols, carboxylic acids, sulfhydryl compounds and amines. Likewise, conjugation with glutathione is another important Phase II reaction that renders highly toxic metabolites harmless. Conjugation of these intermediates with glutathione prevents binding with the nucleic acids, therefore preventing the occurrence of mutations. Metabolites of organic solvents such as benzene, chloroform, and carbon tetrachloride are conjugated with glutathione resulting in decreased toxicity.

The role of the toxic intermediate produced during biotransformation reaction and its effect on the body depends on the rate at which the intermediate undergoes further metabolism to less toxic substances, rate at which it is produced and accumulated in cells, the type of cellular damage caused by the toxic intermediate, and the factors that affect excretion of the toxic material. Phase I and Phase II reactions are designed to deactivate toxic substances by increasing water solubility and excretion of toxic substances. However, these reactions may also bioactivate some toxicants by transforming inactive non-toxic molecules to active toxic forms.

Phase II reactions lead to

Usually inactivation of drug
Production of water soluble metabolites, which is the main aim of biotransformation.
Usually detoxification reaction for xenobiotics (as some substances might be carcinogenic)
There are some important exception, toxic metabolites may be produced by their activation, e.g. methanol is converted into formaldehyde, which is toxic.
Certain conjugation reactions may lead to the formation of reactive species responsible for the hepatotoxicity of drugs.
Products produced might be more potent e.g. M-6-P.

Phase II reactions lead to

Usually inactivation of drug
Production of water soluble metabolites, which is the main aim of biotransformation.
Usually detoxification reaction for xenobiotics (as some substances might be carcinogenic)
There are some important exception, toxic metabolites may be produced by their activation, e.g. methanol is converted into formaldehyde, which is toxic.
Certain conjugation reactions may lead to the formation of reactive species responsible for the hepatotoxicity of drugs.
Products produced might be more potent e.g. M-6-P.

Phase II reactions lead to

Usually inactivation of drug
Production of water soluble metabolites, which is the main aim of biotransformation.
Usually detoxification reaction for xenobiotics (as some substances might be carcinogenic)
There are some important exception, toxic metabolites may be produced by their activation, e.g. methanol is converted into formaldehyde, which is toxic.
Certain conjugation reactions may lead to the formation of reactive species responsible for the hepatotoxicity of drugs.
Products produced might be more potent e.g. M-6-P.

Reactions are mainly conjugate reactions. Normally phase I reactions are followed by phase II reactions but in case of isoniazid (antituberculous drug), phase II reactions occur before phase I reactions because N-acetyl moiety acts as an endogenous substrate for the conjugation reaction. This is followed by phase I hydrolysis to isonicotinic acid.

Effects of Phase II reactions on drugs

Lipid solubility is totally converted into solubility.
Drugs are generally inactivated
Sometimes drugs are activated e.g. minoxidil is activated to minoxidil-o-sulphate (a vasodilator). Morphine is activated to morphine-6-glucuronide.

Phase III reactions:

Some reactions coupled with excretory pumps in the cell membranes are known as phase III reactions.

Drugs undergoing these reactions, is still the subject of extensive research even today.

Table 1 Illustrates the enzymes that catalyse the Phase I and Phase II biotransformation reaction and location where the reaction occurs in the body.

Summary

To summarize, at the end of this module we have studied about

biotransformation
enzymes involved in biotransformation
properties and distribution of enzymes involved in biotransformation
stereochemical aspects of biotransformation
enzymatic reactions that are involved in biotransformation

you can view video on Biotransformationby enzymes

References

Kieslich K (1984) Biotechnology. A Comprehensive treatise in 8 volumes. Rehm H-J and Reed G. (eds.) Biotransformation 6: 1.
Lilly MD (1984) Advances in biotransformation processes. Trans Inst Chem Eng 72: 27-34
Andrew Parkinson, Biotransformaton of xenobiotics