Classification of Enzymes 1 Oxidoreductases 2 Transferases 3

Classification of Enzymes 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases

Proteases Specificity Endoproteases Exoproteases Catalytic mechanism Serine protease Cysteine proteases nucleophilic catalysis Aspartyl proteases acid-base catalysis Metal proteases electrophilic catalysis

HIV Protease ASP 25’

Pepsin ASP 215 ASP 32

Aspartyl Proteases • Two Asp residues in the catalytic site • The two residue can be on the same chain or on different chains • Optimum p. H is acidic: HIV-PR p. H 4 -5; pepsin p. H ≈ 4 (stomach) 1 st p. Ka: 3. 3 2 nd p. Ka: 5. 3 1 st p. Ka: 4. 21 2 nd p. Ka: 5. 64

HIV-Protease – Catalytic Mechanism Tetrahedral Intermediate (hydrated amide)

Brønsted Equation Is there a relation between KHA and k. HA (KHB and k. HB)? The Brønsted equation (empirical) log k. HA = alog. KHA + cost. log k. HA = -ap. KHA + cost. (0 ≤ a ≤ 1) logk. B = -blog. KBH + cost. log k. B = bp. KB + cost. (0 ≤ b ≤ 1)

Brønsted Equation LFER = Linear Free Energy Relationship log k. HA = a log KHA + cost

Brønsted Equation Meaning of the a, b parameters DG’# - DG# = a(DG’ – DG) DDG# = a. DDG a = DDG# DDG

Brønsted Equation Halogenation of Carbonyl Compounds v = k. OH[OH-][acetone] + k. B[B][acetone] slow v = k 1[B][acetone] fast Hammond Postulate log k. B G b=0. 48 b=0. 88 p. KBH+ q

Brønsted Equation: Levelling of a (b) log k. B log k. A b=0 a >0 b >0 p. KAH Kinetic effect k. HA becomes faster as HA becomes stronger Thermodynamic effect As HA becomes stronger [HA] << [H 3 O+] (strong acids in water are all the same) p. KBH+ Diffusion rate Reactions in solution can not be faster than diffusion limit: kdiff ≈ 1010 M-1 s-1