Cytochromes P450 Notes

Cytochromes P450

Introduction to Cytochromes P450

• Cytochromes P450 are a crucial family of enzymes involved in phase I metabolism.

• They are located in the endoplasmic reticulum and mitochondria of eukaryotic cells.

• Each cytochrome P450 enzyme contains a single haem molecule as a prosthetic group.

• When reduced and bound to carbon monoxide (CO), they form a complex that exhibits maximum absorbance at 450 nm.

Nomenclature

• The nomenclature of cytochrome P450 enzymes follows a specific format:

  • Root: CYP

  • Family: A number indicating the family (e.g., CYP1, CYP2, CYP3). Mammalian families have less than 40% sequence similarity between them.

  • Subfamily: A letter designating the subfamily (e.g., 2A to 2W). Members within a subfamily show 40-70% sequence similarity.

  • Number: An individual number representing an isoform, with 70-95% sequence similarity between different isoforms (e.g., CYP1A1, CYP1A2).

  • Allelic variants: Represented by >95% homology and products of the same gene (e.g. CYP2D63, CYP2D64).

History of Study

• Prior to 1960:

  • It was known that the liver endoplasmic reticulum was the primary site for xenobiotic metabolism.

  • The process required NADPH and O2.

  • Researchers suspected the involvement of one or two enzymes.

• 1960 to 1980:

  • Cytochrome P450 was identified spectroscopically by the peak at 450 nm when reduced and bound to CO.

  • Evidence suggested the existence of multiple forms of the enzyme.

  • Reconstitution experiments were conducted.

• 1980 onwards:

  • Proteins were purified, and genes were cloned.

  • At least 50 different human P450s are now known.

• 2000 onwards:

  • X-ray structures of human P450s were determined.

Evolution

• Cytochromes P450 have evolved over a long period:

  • 1.5 billion years ago

  • 400 million years ago

  • Today

Functions

• Cytochromes P450 have several key functions:

  • Xenobiotic metabolism: Primarily carried out by families 1, 2, and 3, mainly in the endoplasmic reticulum.

  • Steroid, fatty acid, and vitamin oxidation: Various enzyme isoforms are involved, including:

    • CYP4 for fatty acid oxidation

    • CYP26 for retinoic acid oxidation

    • Mainly occurs in endoplasmic reticulum

  • Steroid biosynthesis: Cytochromes P450 are found in mitochondria and are involved in the biosynthesis of steroids from cholesterol. For example, aromatase synthesizes estrogen.

Mammalian P450 Families

Biochemical Properties

• Cytochromes P450 are monomeric proteins with a molecular weight ranging from 40,000 to 50,000.

• They all contain haem as a prosthetic group.

• The iron atom in the haem is coordinated to the four nitrogens of the haem tetrapyrrole ring and to a cysteine residue at the C-terminal end of the protein.

• Iron is in the Fe(III) state.

• The sixth coordination position is generally occupied by water in the absence of substrate binding.

• Regions of the protein involved in haem and oxygen binding are typically conserved, but substrate-binding areas vary more in sequence.

• Crystal structures of most human P450s relevant to xenobiotic metabolism are now available.

Protoporphyrin IX

• Iron $(Fe^{3+})$ coordinates with 4 nitrogens.

• Also has a link to Cys on P450 protein, and the 6th coordination point is normally water.

Conserved Areas

• Conserved Domains

  • Membrane insertion segment

  • Cytochrome b5 binding

  • Substrate specificity

  • Oxygen binding site

  • Phosphorylation site

  • Controls regioselectivity for steroid hydroxylation

  • Binding with NADPH-cytochrome P450 reductase

  • Essential Cys in haem-binding domain

Cytochrome P450 Reaction

• The reaction is as follows:

  • $DH + NADPH + O<em>2 \rightarrow DOH + NADP^+ + H</em>2O$

  • DH is a drug

  • DOH is the hydroxylated drug

• Three components are needed:

  • Cytochrome P450 enzyme

  • NADPH-cytochrome P450 reductase

  • Phosphatidylcholine

• NADPH provides 2 protons and 2 electrons in an electron transfer process.

NADPH-Cytochrome P450 Reductase

• NADPH-cytochrome P450 reductase is an essential enzyme in the electron transfer process of the cytochrome P450 reaction in the endoplasmic reticulum.

• It is a flavoprotein that contains flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) as prosthetic groups.

• It interacts with the cytochrome P450 enzyme through electrostatic interactions involving carboxyl groups on the reductase and amino groups (likely lysine residues) on the cytochrome P450.

• Mitochondrial P450s use a different protein called adrenodoxin for electron transfer.

Reaction Mechanism

• The reaction mechanism of cytochrome P450 involves several steps, including the binding of substrate (D), the reduction of iron, and the incorporation of oxygen.

• Simplified representation:

  • $P450-Fe^{3+} + D \rightarrow P450-Fe^{3+}-D$

  • $P450-Fe^{3+}-D + e^- \rightarrow P450-Fe^{2+}-D$

  • $P450-Fe^{2+}-D + O<em>2 \rightarrow P450-Fe^{2+}-O</em>2-D$

  • $P450-Fe^{2+}-O<em>2-D + e^- + 2H^+ \rightarrow P450-Fe^{3+}-O-D + H</em>2O$

  • $P450-Fe^{3+}-O-D + H^+ \rightarrow P450-Fe^{3+} + D-OH$

Flavins in Oxidoreductase

• Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) are essential components of the oxidoreductase.

• These flavins can exist in oxidized, half-reduced (semiquinone), and fully reduced forms, facilitating electron transfer.

Electron Transport

• Electrons are transported from NADPH to cytochrome P450 via FAD and FMN in the reductase.

Induction of Cytochrome P450

• Many, but not all, P450 isoforms can be induced by exposure to certain xenobiotics.

• Induction involves an increase in the amount of mRNA and protein present.

• Different inducers can induce particular isoforms.

• Inducers include polycyclic aromatic hydrocarbons (PAH) and barbiturates.

Effects of Cytochrome P450-Mediated Reactions

• Cytochromes P450 inactivate many drugs, increasing their rate of excretion.

• However, they can also:

  • Activate carcinogens (e.g., benzo[a]pyrene in tobacco smoke becomes carcinogenic after P450-mediated reactions).

  • Produce toxic molecules from harmless drugs (e.g., paracetamol).

  • Activate prodrugs (e.g., cyclophosphamide).

Tissue Distribution

• Cytochromes P450 are found at the highest levels in the liver.

• They are detectable but generally at lower levels in the kidney, lung, intestine, adrenals, and brain.

• Some forms are detected mainly in extrahepatic tissue (e.g., CYP1A1, some steroid biosynthetic P450s).

Introduction to Cytochromes P450

• Cytochromes P450 (CYPs) are a vast and diverse family of enzymes that play a vital role in phase I metabolism, primarily responsible for the biotransformation of numerous xenobiotics and endogenous compounds.

• Located predominantly in the smooth endoplasmic reticulum of eukaryotic cells, particularly in the liver, and also present in mitochondria of some tissues, these enzymes facilitate various biochemical reactions.

• Each cytochrome P450 enzyme contains a single haem molecule as a prosthetic group, which is essential for its enzymatic function.

• Upon reduction and when bound to carbon monoxide (CO), they form a complex that exhibits a distinct peak with maximum absorbance at 450 nm, utilized in their identification and characterization.

Nomenclature

• The nomenclature of cytochrome P450 enzymes follows a systematic format:

  • Root: CYP, denoting cytochromes P450.

  • Family: A number indicating the family, such as CYP1, CYP2, or CYP3, with mammalian families exhibiting less than 40% sequence similarity.

  • Subfamily: A letter designating the subfamily (e.g., 2A to 2W). Members within a subfamily demonstrate 40-70% sequence similarity, indicating closely related functions.

  • Number: An individual number representing a specific isoform, with 70-95% sequence similarity observed among different isoforms (e.g., CYP1A1, CYP1A2).

  • Allelic Variants: Represented by symbols indicating variants that show >95% homology and are produced by the same gene (e.g., CYP2D63, CYP2D64), which can affect drug metabolism and efficacy.

History of Study

• Prior to 1960:

  • It was understood that the liver endoplasmic reticulum was the primary site for the metabolism of xenobiotics. Researchers identified the need for cofactors such as NADPH and O2 in these metabolic processes, suspecting the involvement of one or two key enzymes.

• 1960 to 1980:

  • The cytochrome P450 family was defined spectroscopically by the characteristic absorbance peak at 450 nm when the enzyme was reduced and bound to carbon monoxide. Evidence accumulating during this period suggested the existence of multiple enzyme forms.

  • Reconstitution experiments provided further insights into the diverse interactions and variations among these enzymes.

• 1980 onwards:

  • Advances in purification techniques allowed for the purification of various cytochrome P450 proteins, leading to the cloning of their corresponding genes. As a result, the identification of at least 50 distinct human P450 enzymes was achieved.

• 2000 onwards:

  • The structural biology of cytochromes P450 advanced significantly with the determination of their x-ray crystal structures, facilitating a greater understanding of their mechanisms and functions at the molecular level.

Evolution

• Cytochromes P450 have undergone significant evolutionary changes over a long period, estimated at about 1.5 billion years, yielding a complex family of enzymes equipped to handle diverse substrates—from primordial compounds to complex modern synthetic drugs. Their evolutionary adaptability showcases their critical role in the survival of organisms facing various pharmacological and environmental challenges.

Functions

• Cytochromes P450 are involved in several critical metabolic functions:

  • Xenobiotic Metabolism: Primarily mediated by families 1, 2, and 3, these enzymes facilitate the metabolic transformation and detoxification of foreign compounds, including drugs.

  • Steroid and Fatty Acid Oxidation: Multiple enzyme isoforms are engaged in the oxidation processes, including:

  • CYP4 family enzymes primarily responsible for fatty acid oxidation.

  • CYP26 family involved in retinoic acid oxidation, crucial for developmental processes.

  • This oxidative metabolism mainly takes place in the endoplasmic reticulum.

  • Steroid Biosynthesis: Certain cytochromes P450 are localized in mitochondria where they participate in steroid biosynthesis from cholesterol. For instance, aromatase (CYP19) catalyzes the synthesis of estrogens, pivotal for reproductive function.

Mammalian P450 Families

Biochemical Properties

• Cytochromes P450 are monomeric proteins with a molecular weight ranging from 40,000 to 50,000 Dalton, characterized by the presence of haem as a crucial prosthetic group.

• The iron atom in the haem group is tetracoordinated to the four nitrogen atoms of the haem tetrapyrrole ring, in addition to binding a cysteine residue at the C-terminal end of the protein. The iron typically exists in the Fe(III) oxidation state.

• In the absence of substrate binding, the sixth coordination position is generally filled by a water molecule, but this can change upon substrate interaction.

• While the regions of the protein that bind haem and oxygen are highly conserved, the substrate-binding areas demonstrate greater variability in their sequences, allowing for substrate specificity.

• Currently, crystal structures for most human P450s involved in xenobiotic metabolism are available, facilitating drug design and toxicity predictions.

Protoporphyrin IX

• The iron $(Fe^{3+})$ in protoporphyrin IX coordinates with four nitrogens, linking additionally to the cysteine on the P450 protein. The sixth coordination site is typically occupied by water, emphasizing the coordination chemistry that underlies enzyme function.

Conserved Areas

• Conserved Domains

  • Several conserved domains across cytochromes P450 include:

  • Membrane insertion segment, essential for proper localization.

  • Cytochrome b5 binding site, influencing enzyme activity.

  • Substrate specificity region, critical for metabolic function.

  • Oxygen binding site, vital for the catalytic mechanism.

  • Phosphorylation site influencing regulatory control.

  • Controls regioselectivity for steroid hydroxylation, determining product outcomes.

  • Binding with NADPH-cytochrome P450 reductase, which is crucial for electron transfer.

  • An essential cysteine in the haem-binding domain required for catalytic activity.

Cytochrome P450 Reaction

• The enzymatic reaction catalyzed by cytochrome P450 can be summarized as follows:

  • $DH + NADPH + O<em>{2} ightarrow DOH + NADP^{+} + H</em>{2}O$

  • Where DH represents the drug substrate and DOH denotes the hydroxylated product.

• The reaction requires three integral components:

  • Cytochrome P450 enzyme itself.

  • NADPH-cytochrome P450 reductase, which functions in electron transfer.

  • Phosphatidylcholine, a phospholipid that helps stabilize the enzyme complex.

• NADPH provides two protons and two electrons during the redox reaction, essential for the transformation of substrates.

NADPH-Cytochrome P450 Reductase

• NADPH-cytochrome P450 reductase serves as a necessary enzyme in the electron transfer process associated with the cytochrome P450 reaction, operating within the endoplasmic reticulum.

• This flavoprotein contains flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) as prosthetic groups, which are critical for its function.

• The interaction with the cytochrome P450 enzyme occurs through electrostatic forces between carboxyl groups on the reductase and amino groups, likely involving lysine residues within the cytochrome P450 structure.

• It is noteworthy that mitochondrial P450s utilize a distinct protein, adrenodoxin, for electron transfer, highlighting the diversity of electron transport mechanisms in different cellular compartments.

Reaction Mechanism

• The enzymatic mechanism of cytochrome P450 involves multiple sequential steps, including:

  • The binding of the substrate molecule (D).

  • The subsequent reduction of the iron atom.

  • The incorporation of molecular oxygen.

• Simplified representation of the reaction steps:

  • $P450-Fe^{3+} + D <br>ightarrow P450-Fe^{3+}-D$

  • $P450-Fe^{3+}-D + e^{-} <br>ightarrow P450-Fe^{2+}-D$

  • $P450-Fe^{2+}-D + O<em>{2} ightarrow P450-Fe^{2+}-O</em>{2}-D$

  • $P450-Fe^{2+}-O<em>{2}-D + e^{-} + 2H^{+} ightarrow P450-Fe^{3+}-O-D + H</em>{2}O$

  • $P450-Fe^{3+}-O-D + H^{+} <br>ightarrow P450-Fe^{3+} + D-OH$

Flavins in Oxidoreductase

• Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential components of the oxidoreductase system within the cytochrome P450 reaction, functioning as electron carriers.

• These flavins can exist in multiple oxidation states, including oxidized, half-reduced (semiquinone), and fully reduced forms, facilitating efficient electron transfer during enzymatic reactions.

Electron Transport

• Electrons are transferred from NADPH to cytochrome P450 through the intermediary flavins (FAD and FMN) present in NADPH-cytochrome P450 reductase, ensuring a rapid and efficient transfer of reducing equivalents necessary for substrate oxidation.

Induction of Cytochrome P450

• A notable characteristic of many, though not all, cytochrome P450 isoforms is their capacity to be induced by specific xenobiotics. This induction results in an increase in mRNA levels and corresponding proteins, enhancing the metabolic capability of the enzymes.

• Different inducers can selectively promote the expression of particular isoforms, which is critical in pharmacology for understanding drug interactions and variability in drug metabolism among individuals.

• Common inducers include polycyclic aromatic hydrocarbons (PAHs) and barbiturates, which can significantly affect the metabolism of co-administered drugs.

Effects of Cytochrome P450-Mediated Reactions

• Cytochromes P450 play a dual role in drug metabolism; they can both activate and inactivate drugs, influencing their pharmacokinetics significantly:

  • Many drugs are rendered inactive by P450-mediated metabolism, increasing their rate of excretion from the body.

  • However, these enzymes can also activate carcinogens, such as benzo[a]pyrene found in tobacco smoke, converting them into more harmful forms.

  • Additionally, they may convert innocuous compounds into toxic metabolites, illustrated by the metabolic pathway of paracetamol (acetaminophen).

  • Conversely, P450s can also activate prodrugs, such as cyclophosphamide, which require metabolic activation to exert their therapeutic effects.

Tissue Distribution

• The tissue distribution of cytochromes P450 is variable, with the highest concentrations found in the liver, reflecting its central role in drug and xenobiotic metabolism.

• They are present, albeit at lower levels, in other tissues like the kidney, lung, intestine, adrenals, and brain; some isoforms are specifically localized to extrahepatic tissues, such as CYP1A1 which is essential for bioactivation of certain carcinogens, highlighting the diverse roles P450 enzymes play across different body systems.