P450 CYCLE All P450s follow the same catalytic cycle of; 1. Initial substrate binding 2. First electron reduction 3. Oxygen binding 4. Second electron transfer 5 and 6. Proton transfer/dioxygen cleavage

• P450s contain a range of conserved structural elements needed for the

common processes of enzyme reduction and dioxygen binding and activation.

• Substrate selectivity is the result of numerous hypervariable regions distributed throughout the protein that make up the substrate binding cavity.

Global Features 1. Microsomal P450s – typically metabolize drugs and other xenobiotics 2. Cytosolic P450s – soluble bacterial forms with tight substrate

specificities 3. Mitochondrial P450s – typically metabolize steroids (endogenous

compounds)

1. 2. 3.

Structural Features of Cytochrome P450s – COMMON FOLD • Typical ‘heart shaped’ P450 protein • b-sheet-rich (copper arrows) N-terminus; alpha-helix-rich (blue coils)

C-terminus • Active site area above the heme bounded by I-helix and F-G domain • Substrate binding cavity shown in green. • Heme center (red) buried in lipophilic core between I- and L -helices

• F-G helix domain lies perpendicular to I-helix and forms a ‘lid’ to the

active site which lies distal to the heme

HEME BINDING POCKET

• P450 signature sequence - FxxGxxxCxG

• Propionate groups neutralized by hydrogen bonding to Arg, His, Tyr

residues • Methyl and vinyl side-chains intercalated between hydrophobic residues • Several CYP4 enzymes have the unusual property of a covalently-bound

heme that is attached to the protein backbone by an ester link between an I-helix glutamate and the C-5 methyl group of the heme

OXYGEN ACTIVATION Davydov et al., JACS 123:1403-1415 (2001), Vaz et al., PNAS 93:4644-8 (1996); PNAS 95:3555-60 (1998) • Formation of P450 Compound I (Nature’s blowtorch) requires

ordered proton transfer. • The input of protons is controlled by the proton transfer groove, a

highly conserved area of the I-helix immediately adjacent to the heme iron.

• Unified motif for proton transfer groove is; {ASTG}{ASTG}{DEQN}T{ASTG}

High valent iron-oxo species, FeV=O; Porphyrin radical cation

FeV

O

S

FeIV

O

S

FeIV

O

S

• Of special importance is the invariant Thr residue and an adjacent

Acid/Amide residue (sometime referred to as the alcohol-acid pair, whic together funnel protons to the external oxygen of the O-O bond to catalyze heterolytic scission and formation of P450 Compound I.

• Although this has been well worked out for the soluble bacterial

P450, P450 cam, it is less clearly established in mammalian P450s.

• Nonetheless, a common device used by researchers working with animal and human forms of P450 is to mutate the conserved Thr to an Ala, e.g. T302A in CYP2B4, making the assumption that the P450 cycle will be backed up in the mutant so that the at any time there will be a lower concentration of the high valent iron-oxo species and increased levels of the hydroperoxo-iron precursor(s).

• Often this has been used to make conclusions about what types of reactions are catalyzed by which different forms of activated P450.

H H ---- Thr252

O

H

O H ---- Asp251

(S-ligation) Proximal

Distal

FeIII

Cys357

O O

P450 REDUCTION

• Two electrons are provided by the cofactor NADPH.

• Electrons are transferred to the enzyme, one at a time, via the co-enzyme cytochrome P450 reductase.

• In some cases, cytochrome b5 inputs the second electron.

• Enzymes all have a largely cytosolic topology.

• Both co-enzymes appear to interact with P450 via electrostatic interactions with basic residues on the proximal face of the P450; the C-D helices and the K-helix, primarily.

Membrane Orientation, Substrate Access and Product Release • As noted earlier, nearly all P450s sit on top of ER lipid bilayer, facing

the cytoplasm . • Only the hydrophobic N-terminus is ‘buried’ in the membrane. • The F-G domain also contacts the membrane.

• The F-G domain together with the B-C loop appear to be the most likely ‘gates’ to substrate access from the membrane.

• However, multiple ingress/egress channels into the cytosol may exist. (Shah et al., Biochemistry 51:7225 (2012).

• N.B. CYP2W1 – a glycosylated, colon tumor-specific displays the opposite (lumenal) orientation! Gomez et al., Mol. Pharmacol (2010).

SUBSTRATE BINDING HowdoP450sdiscriminatebetweendifferentligands? • Clearly, physiochemical features of the ligand play an important role in

determining P450 isoform specificity. • A ligand will show high selectivity for a particular P450 isoform when

the ligand’s size, shape and charge are complementary to the active site features of that particular P450.

CYP2B6

CYP2C9

CYP2E1 CYP2A6

VOLUME

pKa

PLANARITY High Low

Medium

CYP2C19

CYP1A2

Basic CYP2D6 Acidic

High Low CYP3A4 CYP2C8

P450 P450

P450 P450 P450

P450

All of the most important human drug metabolizing P450s have now been crystallized, providing insights into their ligand selectivities. The ligand-binding cavities of the various P450s vary substantially in size from as small as 190 cubic angstroms (2E1) to > 1500 cubic angstroms (3A4, 2C8). It is important to note that these P450s display a high degree of plasticity, meaning that the active site can deform to accommodate diverse ligands, even within a given P450 isoform.

CYP3A4 Williams et al., Science 305:683-6 (2004) Yano et al., JBC 38091-4 (2004) Ekroos and Sjogren, PNAS 103: 13682 (2006) • CYP3A4 has the widest substrate specificity of the human P450s,

capable of metabolizing large, hydrophobic molecules.

• The enzyme’s crystal structure shows that CYP3A4 has ample space available directly above heme rings, a short F-helix, and a large F-F’ loop featuring an extended aromatic (Phe) cluster.

• The enzyme was initially crystallized with metyrapone bound in the active site and with progesterone bound at a peripheral site above the Phe cluster.

• The enzyme has also been crystallized with 2 molecules of ketoconazole in the active site, and with erythromycin bound in multiple orientations (see Atkins lecture on Activation later in the course).

CYP2C8 Schoch et al., JBC 279: 9497-503 (2004), Schoch et al., JBC (2008).

• The active site of CYP2C8 is large, like CYP3A4, but more sinuous and has an overall Y-shape that can accommodate anionic ligands in one of the distal arms.

• CYP2C8 has been crystallized with several other large ligands and with

2 molecules of 9-cis retinoic acid in the active site, demonstrating much conformational flexibility.

• The enzyme appears to exist as a dimer in ER membranes (Hu et al., DMD, 2010).

CYP2C9 (Williams et al., Nature 424 464(2003); Wester et al., JBC 279:35630 (2004), Maekwa et al., Biochemistry 2017) S-warfarin structure

• Active site is ‘mushroom’ shaped and has sufficient volume to

accommodate multiple ligands.

• Available crystal structures with S-warfarin and flurbiprofen [both weak acids] bound indicate different orientations.

• Flurbiprofen is bound much closer to the heme (~4 angstroms vs 10 angstroms for S-warfarin).

• This S-warfarin orientation is unlikely to be the catalytically productive one.

• Instead, S-warfsrin may be bound in an ancillary binding pocket possibly related to allosterism that is evident for this (and other) P450(s) – see Atkins lecture on Activation later in the course.

• Many other structures are now available, including a recent one with losartan bound at three different sites – peripheral, access channel and in the active site.

Flurbiprofen structure

Losartan structures

• A critical interaction for flurbiprofen is the charge-pairing between R108 on the flexible B-C loop and the carboxylic acid of this NSAID.

• Also aromatic interactions with

F114 and F476 are important for CYP2C9 catalysis of both flurbiprofen and S-warfarin (Mosher et al., Biochemistry, 2009).

CYP2C19 (Reynald et al., JBC, 2012)

• CYP2C19 has been crystallized with OXV bound. • OXV is a benzbromarone derivative (potent CYP2C9 inhibitor) that has

had its halogens replaced with methyl groups, thereby changing the pKa substantially from ~5 to ~8.

• OXV exhibits a Ki for CYP2C19 of ~35 nM.

CYP2D6 • Remember CYP2D6 prefers basic substrates – essentially the opposite of

CYP2C9. • Early visual inspection of CYP2D6 substrates showed that the distances

from the amine moiety to the site of oxidation were almost always 5 -7 Å. • Mutagenesis studies have implicated Asp301, Glu216, Phe120 and Phe483

in substrate binding. Enzyme was first crystallized in the absence of a ligand (Rowland et al., JBC 281:7614, 2006). Debrisoquine modeled into the structure is shown below, first in an early docking (non-catalytic) orientation where Glu216 and Phe 483 interact with the molecule.

• Modeling of debrisoquine into this active site - in a catalytically productive orientation, suggested subsequent critical interactions with Phe120 and Asp301.

N

NH

NH2 N

NH

NH2

OH

• Recently, structures have been solved with a prototypical substrate

(thioridazine) Wang et al., JBC 290: 5092 (2015).

• One structure revealed two molecules of thioridazine within the active site. The molecule poised for oxidation on the tricyclic ring was stabilized by a charge-pair interaction between Asp301 and the protonated nitrogen on the piperidine ring. The second molecule was stabilized at this position by Glu222, which may serve as the ‘bait’ for substrates approaching the active oxygen species.

CYP1A2 Sansen et al., JBC 282:14348 (2007).

• Like CYP1A1, prefers planar, aromatic molecules e.g. polycyclic

aromatic hydrocarbons such as a-naphthoflavone shown below.

• CYP1A2 has a small active site, but one which is well adapted to the

binding of planar aromatic molecules.

CYP2E1 Porubsky et al., J Biol Chem 283:33698 (2008) • Prefers small, solvent-like molecules, but can also metabolize larger fatty

acids. • 4-Methyl pyrazole is an inhibitor that is bound at the heme by an

electrostatic interaction with Thr 303 (B) • Upon binding of larger fatty acids, Phe 298 rotates allowing the samll

active site cavity to merge with a larger adjacent void to accommodate the longer chain compounds, shown in C as w-imidazoyl decanoic acid.

N

HN

H3C

CH3 CH2OH

CO2H

Phe298

CYP2A6 Yano et al. Nature Structural and Molecular Biology 12(9):822 (2005) • Prefers small, often bicyclic, molecules, e.g. nicotine, coumarin

• Crystal structure shows small active site (like CYP1A2), ~25% the volume

of most other mammalian P450s. • Asn297 is important electrostatic anchoring site for coumarin binding.

O O O OHO

N

NCH3

N

NCH3

O

CYP2B6 (Gay et al., 2010; Shah et al., 2012) • Crystallization impeded by low expression and thermal instability of

wild-type constructs. • Available structures are for the K262R/Y226H double mutant. • CYP2B6 has been crystallized with 4-chlorophenylimidazole (red mesh)

and amlodipine (brown mesh) bound, illustrating an active site cavity capable of binding bulky molecules near the heme.

• CYP2B6 demonstrates a very highly ‘plastic’ active site able to bind

molecules with molecular weights ranging from ~80-800 Da with similar high affinity.

• CYP2B6 crystallized recently with a-pinene, a pure hydrocarbon. Structure illustrates how CYP2B6 active site cavity adapts to this small molecule that is bound by van der Waal interactions devoid of electrostatics or heme ligation (Wilderman et al, JACS 135:10433 (2013).

HN

O

O

ONH2

O

OCl