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 Chapter 16 Mechanisms of Enzyme Action
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| tarix | 12.10.2018 | | ölçüsü | 2,48 Mb. | | #73973 |
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Chapter 16 to accompanyBiochemistry, 2/ebyReginald Garrett and Charles Grisham
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 13.13 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
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
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
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPAAll 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 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
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
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? If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form Several novel protease inhibitors are currently marketed 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
Lysozyme Lysozyme hydrolyzes polysaccharide chains and ruptures certain bacterial cells by breaking down the cell wallHen egg white enzyme has 129 residues with four disulfide bondsThe first enzyme whose structure was solved by X-ray crystallography (by David Phillips in 1965)
Substrate Analog Studies Natural substrates are not stable in the active site for structural studiesBut analogs can be used - like (NAG)3This argues for stabilization of a transition state via destabilization (distortion and strain) of the substrate
The Lysozyme Mechanism Studies with 18O-enriched water show that the C1-O bond is cleaved on the substrate between the D and E sitesThis incorporates 18O into C1Glu35 acts as a general acidAsp52 stabilizes a carbonium ion intermediate (see Figure 16.37)
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!
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