PHASE I Metabolism:

Many types of reactions. Most result in addition of single atom of oxygen as hydroxyl, ketone, or epoxide.

I. Microsomal monooxygenation:

• Enzymes bound to E.R.
• Addition of single atom of oxygen
• NADPH - dependent

Two systems:

A. FAD-containing monooxygenases:

Add oxygen to amines, sulfur compounds and organophosphorus compounds

B. Cytochrome P-450s

Named for "cytochrome" (i.e. heme) functional group in enzyme

"Mixed-function oxygenases" (MFOs) -- "mixed function"= 1/2 used to oxidize xenobiotic, 1/2 reduced to water.

Large assemblage of > 200 enzymes divided into 28 Families and many Subfamiles:

Nomenclature:

• Gene/gene product: CYP
• Family: 1 - 28 (arabic or roman numerals)
• Subfamily: A - Z (letters)
• Individuals: 1 - ?? (arabic numerals)
• (EG. CYP1A1 or P4501A1)

Family -- >=40% a.a. similarity put into same family

Subfamily -- >=55% a.a similarity put into same subfamily

Individual isozymes -- arbitrarily assigned, based on catalytic specificity

• (up to 97% similarity with other subfamily members)

Used in normal biochemical pathways as well as in xenobiotic metabolism:

Evolutionarily, extremely old group of enzymes, but with widely diverging form and function.

Oldest: 3 billion years -- evolved to maintain membrane integrity

• bacterial steroid & fatty acid metabolism
• now comprise families III and IV (diverged ca. 1bya)

Not long after formation of free molecular oxygen (photosynthetic bacteria&plants), organisms had to deal with oxidative stress.

Biosynthetic and xenobiotic metabolism genes diverged ca. 900 million years ago.

• CYPI and II = xenobiotic metabolism
  • I -- aromatic hydrocarbons; combustion products
  • II -- drugs (e.g., phenobarbital); terrestrial plant 2deg. compounds
• CYPIII = steroid metabolizing
• CYPXI, XVII, XIX, and XXI = steroid biosynthetic pathways

Will concentrate on CYPI and CYPII

Why did they (CYPI and CYPII) evolve?

1) Plant v. Animal warfare

In response to attack by microbes and animal predators, plants evolved a broad class of low-molecular weight stress proteins, generally referred to as phytoalexins (>10,000 known).

• Toxic
• Carcinogenic

Interesting to note that CYPI and CYPII diverged around same time as movement of vertebrates onto land, but that fish do not possess CYPII enzyme activity.

2) Combustion products of organic material (e.g., fossil fuels)

Locations in body: Liver**, lung, brain, skin, nasal mucosa, GI tract

Microsomal (i.e., embedded in endoplasmic reticulum)

• In membrane in close association with:
  • reductase enzyme (NAD(P)H-dependent, flavoprotein)
  • phospholipid (joins two together)
• Substrate must be able to penetrate into membrane to reach active site (i.e., lipophilic)

General reaction of monooxygenase:

Some of the details:

Additional Reactions Within the Catalytic Cycle:

• Interruption of the P450 catalytic cycle at a variety of points can result in the formation of active oxygen species.
• Presence of peroxides can support catalytic cycle in absence of reducing co-factors, via the "peroxide shunt".

Different enzymes are under genetic control and therefore can be INDUCED: Pre-exposure causes increased production

REACTIONS OF MICROSOMES:

1. Epoxidation

Aliphatic or Aromatic

2. Hydroxylation

3. O-, N-, S- Dealkylation --

Dealkyation of ethoxyresorufrin to resorufin is specific biomarker of CYP1A1 activity:

4. Desulfuration (cf. parathion)

II. NON-MICROSOMAL OXIDATIONS:

Located in cytosol or in mitochondria

1. Dehydrogenases:

(cytosolic)

• alcohols ----> aldehydes
• aldehydes ----> acids

2. Amine oxidases:

(mitochondrial)

• endogenous nervous system function -- neurotransmitter turnover
• other tissues -- deamination of aliphatic amines to aldehydes
• byproduct formation of hydrogen peroxide (toxic)

3. Reduction:

(cytosolic)

• addition of H⁺ + e^-

4. Hydrolysis:

(microsomal and cytosolic)

• carboxylesterases and amidases
• eg., ACHase (normal nervous system function)

5. Epoxide hydration:

(microsomal and cytosolic)