Inhibition of enzymatic activity Inhibitors are chemicals that reduce the rate of enzymatic reactions. Activators are chemicals that increase the rate of enzymatic reactions Inhibition may be a. irreversible (=inactivation) b. reversible b1. mixt (most general case) b2. competitive b3. non-competitive b4. un-compétitive

Inhibition of enzymatic activity irréversible inhibition (=inactivation) E + I EI => E-I First step a non-covalent complex, second step a covalente bond) How to distinguish between irreversible and reversible inhibition? 1. Incubation of the enzyme with the inhibitor 2. Dialyse , gel filtration or just dilution 3. Activity measurement. If activity restores, inhibition was reversible Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase

Inactivation of enzymatic activity

PGH = prostaglandin; controls inflamation

Inactivation of enzymatic activity REMEMBER: nucleophiles in proteins NH

KI

E+I

OH

kcat

EI

E- I *

SNH 2

diisopropylphosphofluoridate (DIPF)

Only the active-site serine of serine proteases reacts with DIPF; The DIPF is (relatively) stable in solution Will talk more about this reaction later

N CO2 -

Inactivation of enzymatic activity Organo-phosphoric pesticides inactive the acetyl-cholineasterase

Dose létale: 0.5 mg

Catalytic mechanism of acetyl-cholinesterase

Inactivation of chymotrypsine

TPCK is the irreversible inhibitor of chymotrypsin. It does not inhibit trypsin since chymotrypsin is specific for amino acids having hydrophobic side chains. This reaction is used for the preparation of chymotryptin-free trypsin

Reversible - MIXED inhibition k1

k2

k-1

KI =

[E][I] [EI]

and K' I =

[ES ][I] [ESI]

Mixed inhibition is the most general case. The inhibitor binds to the free enzyme and to the ES complex. The affinities (Kd) are not equal in general

MIXED inhibition

k1

k2

k-1 KI =

[E][I] [EI]

and K' I =

[ES ][I] [ESI]

This equilibrium is not used for deriving the Michaelis equation since it is a function of the three others (a thermodynamic cycle)

MIXED inhibition k1 k-1

As usual: [S]>>[E] and [I]>>[E]; otherwise very complicated equations

k2

MIXED inhibition k k1 -1

k2

MIXED inhibition k k1 -1

α = 1 + [ I ]/KI ; α’ = 1 + [ I ]/K’I Km,app = Km * α / α’ Vmax,app = Vmax / α’ ( Vmax/ Km )app = ( Vmax/ Km ) / α

k2

MIXED inhibition

COMPETITIVE Inhibition k1

k2

k-1

Competitive inhibition: a particular situation where S and I cannot bind Simultaneously to the enzyme

COMPETITIVE Inhibition k1 k-1

α = 1 + [ I ]/KI ; Km,app = Km * α Vmax,app = Vmax ( Vmax/ Km )app = ( Vmax/ Km ) / α

k2

COMPETITIVE Inhibition k1 k-1

k2

COMPETITIVE Inhibition k1 k-1

k2

Examples of COMPETITIVE Inhibition Very often the competitive inhibitors are structural analogs of substrates; but not always!

Fumarate + 2H++ 2e-

Succinate

Succinate dehydrogenase CH2COO-

COO-

CHCOO-

COO-

CHCOO(trans)

CH2 CH2COO-

Malonate

Examples of COMPETITIVE Inhibition

Most drugs are enzyme inhibitors. I will give some examples. The main problem with drugs which are competitive inhibitors is that the presence of substrate decreases the degree of inhibition. A second problem is enzyme inhibition of both normal and pathological tissues. Toxicity is closely linked to therapy. Antibiotics agains bacteria are more easy to develop (but nothing ir really easy in the therapy field)! In fact, bacteria is a procaryotic organisms and often some metabolisms are absent in mammalian, or the enzymes are quite different. Some drugs are not « inhibitors » but active substrate analogs (anti-metabolites)

Sulfamides, the first antibiotics NH2

O

O

NH2

O

S

NHO

Sulfamides, the first antibiotics Inhibitors of the folic acid synthesis Analogs of benzoic acid pKa approx 4

NH2

O

NH2

NH2

OH

pKa approx 3

O pKa approx 4

NH2

O

S

O

pKa approx 6 NH2

O

O

S

NHO O

Allopurinol, inhibiteur de la xanthine oxydase

Gout is a medical condition that usually presents with recurrent attacks of acute inflammatory arthritis (red, tender, hot, swollen joint). It is caused by elevated levels of uric acid in the blood. The uric acid crystallizes and deposits in joints, tendons, and surrounding tissues. Gout affects 1% of Western populations at some point in their lives.The enzyme xanthine oxidase, or XO, (bovine milk enzyme is PDB 1FIQ, EC 1.17.3.2) catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid: hypoxanthine + O2 + H2O xanthine + H2O2 xanthine + O2 + H2O uric acid + H2O2

Methotrexate, inhibitor of dihydrofolate reductase Le methotrexate, a cytostatic (anti-tumor agent) is an analog of dihydrofolate which is necessary for the synthesis of Thymidine nucleotides and therefore for DNA synthesis. Inhibition of Dihydrofolate reductase stops finally DNA synthesis and cell replication.

An useful hint: larger molecules bind more tightly to enzymes ! KD ( M )

Derivative O

Biotin HN

1.3 x 10-15

NH (CH2)4

S

Biotin is tightly bound by avidin, a protein from hen egg. Detailed studies showed that biotin has a stronger affinity than that calculated from the affinity of fragments.

COO-

Why? O

HN

Desthiobiotin

KAB 5 x 10-13 (instead of the calculated value 10-7 M)

NH

H 3C

(CH2)4

COO-

O HN

KB 3.4 x 10-5

NH

H3C H 3C

(CH2)4

COO-

KA 3 x 10-3

KAB