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Chapter 16 Mechanisms of Enzyme Action Outline • • • • • • • • • 13.1 Stabilization of the Transition State 13.2 Enormous Rate Accelerations 13.3 Binding Energy of ES 13.4 Entropy Loss and Destabilization of ES 13.5 Transition States Bind Tightly 13.6 - 13.9 Types of Catalysis 13.11 Serine Proteases 13.12 Aspartic Proteases 13.13 Lysozyme 16.1 Stabilizing the Transition State • Rate acceleration by an enzyme means that the energy barrier between ES and EX* must be smaller than the barrier between S and X* • This means that the enzyme must stabilize the EX* transition state more than it stabilizes ES • See Eq. 16.3 16.2 Large Rate Accelerations See Table 16.1 • Mechanisms of catalysis: • – Entropy loss in ES formation – Destabilization of ES – Covalent catalysis – General acid/base catalysis – Metal ion catalysis – Proximity and orientation 16.3 Binding Energy of ES Competing effects determine the position of ES on the energy scale • Try to mentally decompose the binding effects at the active site into favorable and unfavorable • The binding of S to E must be favorable • But not too favorable! • Km cannot be "too tight" - goal is to make the energy barrier between ES and EX* small 16.4 Entropy Loss and Destabilization of ES Raising the energy of ES raises the rate • For a given energy of EX*, raising the energy of ES will increase the catalyzed rate • This is accomplished by • – a) loss of entropy due to formation of ES – b) destabilization of ES by • strain • distortion • desolvation 16.5 Transition State Analogs Very tight binding to the active site! • The affinity of the enzyme for the transition state may be 10 -15 M! • Can we see anything like that with stable molecules? • Transition state analogs (TSAs) do pretty well! • Proline racemase was the first case • See Figure 16.8 for some good recent cases! 16.6 Covalent Catalysis Serine Proteases are good examples! • Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway • The formation of the covalent bond provides chemistry that speeds the reaction General Acid-base Catalysis Catalysis in which a proton is transferred in the transition state • "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center • "General" acid-base catalysis involves acids and bases other than H+ and OH• These other acids and bases facilitate transfer of H+ in the transition state • See Figure 16.12 The Serine Proteases Trypsin, chymotrypsin, elastase, Thrombin, subtilisin, plasmin, TPA • All involve a serine in catalysis - thus the name • Ser is part of a "catalytic triad" of ser, his, asp • Serine proteases are homologous, but locations of the three crucial residues differ somewhat • Enzymologists agree, however, to number them always as His-57, Asp-102, Ser-195 • Burst kinetics yield a hint of how they work! Serine Protease Mechanism A mixture of covalent and general acid-base catalysis • Asp-102 functions only to orient His-57 • His-57 acts as a general acid and base • Ser-195 forms a covalent bond with peptide to be cleaved • Covalent bond formation turns a trigonal C into a tetrahedral C • The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195 The Aspartic Proteases Pepsin, chymosin, cathepsin D, renin and HIV-1 protease • All involve two Asp residues at the active site • Two Asps work together as general acid-base catalysts • Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry • HIV-1 protease is a homodimer Aspartic Protease Mechanism The pKa values of the Asp residues are crucial • One Asp has a relatively low pKa, other has a relatively high pKa • Deprotonated Asp acts as general base, accepting a proton from HOH, forming OH- in the transition state • Other Asp (general acid) donates a proton, facilitating formation of tetrahedral intermediate Asp Protease Mechanism - II See Figure 16.27 • What evidence exists to support the hypothesis of different pKa values for the two Asp residues? • See the box on page 525 • Bell-shaped curve is a summation of the curves for the two Asp titrations • In pepsin, one Asp has pKa of 1.4, the other 4.3 HIV-1 Protease A novel aspartic protease • HIV-1 protease cleaves the polyprotein products of the HIV genome • This is a remarkable imitation of mammalian aspartic proteases • HIV-1 protease is a homodimer - more genetically economical for the virus • Active site is two-fold symmetric • Two Asp residues - one high pKa, one low pKa Therapy for HIV? Protease inhibitors as AIDS drugs • If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form • Several novel protease inhibitors are currently used as AIDS drugs • Many such inhibitors work in a culture dish • However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body • See the box on page 524! Chapter 16 Problems Work the end-of-chapter problems! • Number 2 is particularly good • Note in the Science article referenced in number 2 that the figure legend has a mistake!