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Biotransforming Enzymes, Substrates and End Products Biotransformation is the process within our body whereby a substance is changed, mainly transformed, from one chemical to another by a chemical reaction. It is also known as Metabolism or metabolic transformations. Biotransformation is vital to survival in that it transforms absorbed nutrients (food, oxygen, etc.) into substances required for normal body functions. For some pharmaceuticals, it is a metabolite that is therapeutic and not the absorbed drug. Like, phenoxybenzamine, a drug given to relieve hypertension, is biotransformed into a metabolite, which is the active agent. It also serves as an important defense mechanism in those toxic xenobiotics and body wastes are converted into less harmful substances and that can be excreted from the body. Biotransforming Enzyme: Chemical reactions are continually taking place in the body. Most of these chemical reactions occur at significant rates only because specific proteins, known as enzymes, are present to catalyze them, that is, accelerate the reaction. These enzymatic reactions are not always simple biochemical reactions. Some enzymes require the presence of cofactors or co-enzymes in addition to the substrate before their catalytic activity can be exerted. Substrate is the substance that will be catalyzed. These co-factors exist as a normal component in most cells and are frequently involved in common reactions to convert nutrients into energy; Vitamins are an example of co-factors. It is the drug or chemical transforming enzymes that hold the key to xenobiotic transformation. The relationship of

substrate, enzyme, co-enzyme, and transformed product is illustrated below: Figure 01: Relationship of substrate, enzyme, co-enzyme, and transformed product Most biotransforming enzymes are high molecular weight proteins, composed of chains of amino acids linked together by peptide bonds. While an enzyme may encounter many different chemicals, only those chemicals (substrates) that fit within the enzymes convoluted structure and spatial arrangement will be locked on and affected. This is sometimes referred to as the "lock and key" relationship. As shown in Figure 1, when a substrate fits into the enzyme's

structure, an enzyme-substrate complex can be formed. This allows the enzyme to react with the substrate with the result that two different products are formed. If the substrate does not fit into the complex will thus no occur. enzyme, no be formed and reaction can

Figure 02: Enzyme-substrates lock and key relationship In mammals there are two categories of enzyme systems are known to exist: Enzymes with normal endogenous substrates Specific Enzymes having no specific endogenous substrates Non-specific

Specific Enzyme: Enzymes that normally occur in the tissues and are responsible for transformation of normal endogenous chemicals in the tissues. Like, Enzyme: Monoamine oxidase (MAO); Substrate: Epinephrine & Norepinephrine, Dopamine, Serotonin, Tyramine. Non-specific Enzyme: Enzymes that alter the structure of many foreign chemicals but have no established normal endogenous substrates. Like: Enzyme: Cholinesterase; Substrate: Acetylcholine, Procaine, Succinylcholine.

Procaine

p-Aminobenzoic acid Figure 03: Metabolism of Procaine

Figure 05: Metabolism of Serotonin The reactions catalyzed by xenobiotic biotransforming enzymes generally are divided into two groups: 1. Phase I reactions Non-synthetic reactions 2. Phase II reactions Synthetic reactions Phase I reaction: Phase I reactions are generally reactions which modify the chemical by adding a functional structure. This allows the substance to "fit" into the Phase II enzyme so that it can become conjugated (joined together) with another substance. In Phase I reactions, a small polar group (containing both positive and negative charges) is either exposed on the toxicant or added to the toxicant. The main Phase I reactions are: Oxidation, Reduction, Hydrolysis.

Table 01: Example of type of reaction and enzymes participate in phase I reaction Reaction Enzyme

Oxidation

Cytochrome P-450 dependent monooxygenase Xanthine oxidase and Xanthine Dehydrogenase Peroxidases Amine oxidase Monoamine oxidase Alcohol Dehydrogenase Aldehyde Dehydrogenase Dihydrodiol Dehydrogenase Molybdenum Hydroxylases Aldehyde Oxidase Monoamine Oxidase Cyclooxygenase Dioxygenases

Reduction

Cytochrome P-450 dependent reductases Ketoreductase Azo Reductase Nitro Reductase Carbonyl Reductase Glutathione peroxidases

Ester hydrolysis

Carboxylesterases Pseudocholinesterases Paraoxonase Peptidases Amidases

Hydration

Epoxide hydrolase

1. Oxidation: Oxidation is a chemical reaction in which a substrate loses electrons. There are a number of reactions that can achieve the removal of electrons from the substrate. The specific oxidizing reactions and oxidizing enzymes are numerous. Here are several of these oxidation reactions.

Alcohol dehydrogenation Aldehyde dehydrogenation Alkyl/acyclic hydroxylation Aromatic hydroxylation Deamination Desulfuration N-dealkylation N-hydroxylation N-oxidation O-dealkylation Sulphoxidation

Table 02: An overview of possible types of phase I biotransformation reaction (oxidations)

Oxidation of Drugs by Cytochrome P450: The cytochrome P450 superfamily (officially abbreviated as CYP) is a large and diverse group of enzymes. The function of most CYP enzymes is to catalyze the oxidation of organic substances. The substrates of CYP enzymes include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for about 75% of the total number of different metabolic reactions. The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g.,

insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water:

RH + O2 + 2H+ + 2e ROH + H2O

Aliphatic Oxidation:

Aromatic Hydroxylation:

N-Dealkylation:

O-Dealkylation:

S-Demethylation:

Oxidative Deamination:

S-Oxidation:

N-Oxidation:

N-Hydroxylation:

Desulfuration:

2. Reduction: Reduction is a chemical reaction in which the substrate gains electrons. Reductions are most likely to occur with xenobiotics in which oxygen content is low. There are fewer specific reduction reactions than oxidizing reactions. Here are several of these reduction reactions. Azo reduction Dehalogenation Disulfide reduction Nitro reduction N-oxide reduction Sulfoxide reduction

Table 03: An overview of possible types of phase I biotransformation reaction (reductions)

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Azo Reduction:

Nitro Reduction:

Dehalogenation: There are three major mechanisms of dehalogenation:

Reductive dehalogenation: Halogen replaced with a hydrogen atom. Oxidative dehalogenation: Halogen & hydrogen replaced with oxygen

iii.

Double dehalogenation: 2 halogens replaced from two adjacent Cs to form a C-C double bond

3. Hydrolysis: Hydrolysis is a chemical reaction in which the addition of water splits the

toxicant into two fragments or smaller molecules. The hydroxyl group (OH-) is incorporated into one fragment and the hydrogen atom is incorporated into the other. Larger chemicals such as esters, amines, hydrazines, and carbamates are generally biotransformed by hydrolysis. Table 04: An overview of possible types of phase I biotransformation reaction (Hydrolysis)

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Ester Hydrolysis:

Epoxide Hydrolase:

Phase II reaction: Phase II reactions consist of those enzymatic reactions that conjugate the modified xenobiotic with another substance. A xenobiotic that has undergone a Phase I reaction is now a new intermediate metabolite that contains a reactive chemical group, e.g., hydroxyl (-

OH), amino (-NH2), and carboxyl (-COOH). Many of these intermediate metabolites do not possess sufficient hydrophilicity to permit elimination from the body. These metabolites must undergo additional biotransformation as a Phase II reaction. Phase II reactions are conjugation reactions, that is, a molecule normally present in the body is added to the reactive site of the Phase I metabolite. The result is a conjugated metabolite that is more water-soluble than the original xenobiotic or Phase I metabolite. Usually the Phase II

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metabolite is quite hydrophilic and can be readily eliminated from the body. The primary Phase II reactions are:

Glucuronide conjugation - most important reaction

Acetylation Amino acid conjugation Glutathione conjugation Methylation

Sulfate conjugation - important reaction

Table 01: Example of type of reaction and enzymes participate in phase I reaction Reaction Methylation Enzyme S-Methyltransferases O- Methyltransferases N- Methyltransferases Acetylation N-Acetyltransferases Acetyltransferases Glucuronide conjugation Glutathione conjugation Glucuronide conjugation: Glucuronosyltransferases Glutathione S-transferase

Amino acid conjugation:

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Methylation:

Acetylation: