Inhibition of enzymatic activity

1. 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

2. 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? • Incubation of the enzyme with the inhibitor • Dialyse , gel filtration or just dilution • 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

3. Inactivation of enzymatic activity PGH = prostaglandin; controls inflamation

4. Inactivation of enzymatic activity REMEMBER: nucleophiles in proteins K k I N c a t E + I E I E- I * N H O H - S N H - 2 C O 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

5. Inactivation of enzymatic activity Organo-phosphoric pesticides inactive the acetyl-cholineasterase Dose létale: 0.5 mg Catalytic mechanism of acetyl-cholinesterase

6. 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

7. Reversible - MIXED inhibition k1 k2 k-1 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

8. MIXED inhibition k1 k2 k-1 This equilibrium is not used for deriving the Michaelis equation since it is a function of the three others (a thermodynamic cycle)

9. MIXED inhibition k1 k2 k-1 As usual: [S]>>[E] and [I]>>[E]; otherwise very complicated equations

10. MIXED inhibition k1 k2 k-1

11. MIXED inhibition k1 k2 k-1 a = 1 + [ I ]/KI ;a’ = 1 + [ I ]/K’I Km,app = Km * a / a’ Vmax,app = Vmax / a’ ( Vmax/Km )app =( Vmax/Km ) / a

12. MIXED inhibition

13. COMPETITIVE Inhibition k1 k2 k-1 Competitive inhibition: a particular situation where S and I cannot bind Simultaneously to the enzyme

14. COMPETITIVE Inhibition k1 k2 k-1 a = 1 + [ I ]/KI ; Km,app = Km * a Vmax,app = Vmax ( Vmax/Km )app =( Vmax/Km ) / a

15. COMPETITIVE Inhibition k1 k2 k-1

16. COMPETITIVE Inhibition k1 k2 k-1

17. Examples of COMPETITIVE Inhibition CH2COO- CHCOO- COO- CH2 CH2COO- CHCOO- (trans) COO- Malonate Very often the competitive inhibitors are structural analogs of substrates; but not always! Fumarate + 2H++ 2e- Succinate Succinate dehydrogenase

18. 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)

19. Sulfamides, the first antibiotics

20. Sulfamides, the first antibiotics Inhibitors of the folic acid synthesis Analogs of benzoic acid pKa approx 4 pKa approx 3 O pKa approx 4 pKa approx 6

21. 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

22. 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.

23. An useful hint: larger molecules bind more tightly to enzymes ! KD ( M ) 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. Why? Derivative Biotin 1.3 x 10-15 Desthiobiotin KAB 5 x 10-13 (instead of the calculated value 10-7 M) KAB << KBxKA (affinity is stronger than expected) KB 3.4 x 10-5 KA 3 x 10-3

24. An useful hint: larger molecules bind more tightly to enzymes ! Desthiobiotin KAB 5 x 10-13 KB 3.4 x 10-5 KA 3 x 10-3 KAB << KBxKA (affinity is stronger than expected)

25. An useful hint: larger molecules bind more tightly to enzymes ! 2 1 2 1 Fragment binding are second-order reactions. Instead, the fusion molecule binds with a second order for the first fragment (2), then as a first order!! The effective concentration describes the favourable effect

26. An useful hint: larger molecules bind more tightly to enzymes ! example: the bisubstrate analogs NMP and ATP are the two substrates of the enzyme Nucleoside monophosphate kinase. A derivative was synthetized where the two substrates are incorporated into the same molecule – dinucleoside pentaphosphate. It strongly binds to the Nucleoside monophosphate kinase dinucleoside pentaphosphate

27. An useful hint: larger molecules bind more tightly to enzymes ! example: the bisubstrate analogs Aspartate and carbamoyl phosphate are the two substrates of the aspartate Carbamoyl transferase. The two moyeties are incorporated in the bisubstrate analog N-(phosphonoacetyl)-L-aspartate Bisubstrate analogs are enormously useful for trapping enzymes in their active conformation.

28. An useful hint: larger molecules bind more tightly to enzymes ! example: some physiological inhibitors are proteins Several protease precursors are present in mammalian plasma (blood). The activated form is active in blood cloting and fibrinolysis. They are a permanent danger. There are in plasma several inhibitors (anti-trypsin, anti-thrombin) at high concentrations, about 1 mg/ml! The inhibitors belong to the SERPIN class (serine protease inhibitors ). They have an unmeasurably high affinity for the target proteases. Each inhibitor molecule inhibits one protease molecule. This is very expensive but necessary for our health.

29. An 2nd useful hint: transition state analogs are strong competitive inhibitors We will talk later on this important topic

30. NON-COMPETITIVE inhibition Inhibiton bounds to the free enzyme and to the ES comples with equal affinity KI = Ki’ a‘ = 1 + [ I ]/KI ; Km,app = Km Vmax,app = Vmax / a’ ( Vmax/Km )app =( Vmax/Km )/ a’ This class of inhibitors does not modify the Km, dut decrease the Vmax and Vmax/Km

31. NON-COMPETITIVE inhibition KI = Ki’ La diminution de l’activité enzymatique n’est pas fonction de la concentration en substrat Favorable pour les médicaments

32. Some anti-AIDS drugs are non-competitive inhibitors of the REVERSE TRANSCRIPTASE PDB 1s1x Nevirapine  potent inhibitor of HIV-RT … KI ~ nM … no resemblance to any of the natural nucleotide substrates … binds in a hydrophobic binding pocket adjacent to the substrate-binding pocket and modifies the rate of polymerization.

33. UN-COMPETITIVE Inhibition The inhibitor binds to the ES comples, but not to the free enzyme. This kind of inhibition is unusual for enzymes with one substrate, but is common for multisubstrate enzymes

34. UN-COMPETITIVE Inhibition a = 1 + [ I ]/KI ; Km,app = Km / a Vmax,app = Vmax / a ( Vmax/Km )app =( Vmax/Km ) Vmax/Km is not affected by un-competitive inhibition. Un-competitive inhibitors are not efficient drugs if the physiological substrate concentration is < Km

35. Excess-substrate inhibition 2 molecules of Acetyl chloline may bind to the acetylcholinesterase active site: one binds with the acetyl, the other with the choline part

36. Excess-substrate inhibition Mg2+ This is common with enzymes having nucleotides as substrates In adenylate kinase, AMP may bind to the ATP site ATP site AMP site

37. Excess-substrate inhibition This is common with enzymes having a ping-pong mechanism Example: the nucleoside diphosphate kinase Mg•N1TP + E-His  Mg•N1DP + E-His-P Mg•N2DP + E-His-P  Mg•N2TP + E-His Sum: Mg•N1TP + Mg•N2DP == Mg•N1DP + Mg•N2TP GDP ATP E-GDP EP-ATP GDP may bind instead of ATP to the free enzyme and is a competitive inhibitor; Similarily, ATP may bind instead of GDP to the phosphorylated enzyme and is a competitive inhibitor aswell

38. Concurrents reactions: it appears sometimes as inhibition! EA  E + P A E B EB  E + Q A and B are both substrates for the enzyme. Our assay allows to follow P, but not Q. B appears to be in this set-up a competitive inhibitor with respect to A. Example: cholinestérase reaction Acetyl-thiocholine, detection with DTNB Acetyl-choline is a substrate, but we cannot « see » it NOTE: This is an easy method for measuring enzyme’s Kd for the substrate (you already know that Km is not the Kd!)

39. Concurrent reactions EA  E + P A E B EB  E + Q A and B are both substrates for the enzyme. Our assay allows to follow P, but not Q. B appears to be in this set-up a competitive inhibitor with respect to A. A clever method to measure the Kd for a bad substrate: both A and B are substrates, but the rate with A is 100-fold or more faster than the reaction with B. Km,app = Km * (1 + [ I ]/KI )

40. Activation of enzyme activity E + S ES No binding EA + S EAS E + products A KA k1 k2 k-1 [E]total = sum of all forms of E [E]total = [E] + [EA] + [ESA] KA [A] [E] KA= ------------ [E] = [EA] ------------ [EA] [A] Common situation: metal-ion activated enzymes (Mg2+, Ca2+)

41. Activation of enzyme activity [E]total = [E] + [EA] + [ESA] KA [A] [E] KA = ------------ [E] = [EA] ------------ [EA] [A] KA [E]total = [EA] -------- + [EA] + [ESA] [A] KA [E]total = [EA] (-------- + 1)+ [ESA] [A]

42. Activation of enzyme activity KA [E]total = [EA] (1+ -------)+ [ESA] [A] X1 [E]total = [EA] X1 + [ESA] Next step: steady-state E + S ES No binding EA + S EAS E + products A KA k1 k2 k-1 k1 [EA] [S] = (k –1 + k 2) [EAS] [EA] = (k –1 + k 2)/ k1 [EAS]/[S]

43. Activation of enzyme activity [E]total = [EA] X1 + [ESA] [EA] = (k –1 + k 2)/ k1 [EAS]/[S] (k –1 + k 2)/ k1 = Km [E]total = [ESA] Km/[S] X1 + [ESA] V = k2 [EAS] k2 [E]total V = --------------------------- Km/[S] X1 + 1 Vmax [S] V = --------------------------- KmX1+ [S]

44. Activation of enzyme activity KA 1 + ------ [A] Vmax [S] V = --------------------------- Km X1+ [S] Vmaxapp[S] V = ----------------- Kmapp + /[S] Vmaxapp = Vmax Vmaxapp 1 Vmax ----- = ------- ------------ Kmapp = Km KA Kmapp 1 + ---- Km [A] [A] is smaller than KA , Km becomes very large