1 ENZYMES Serine Proteases Chymotrypsin, Trypsin, Elastase, Subtisisin Principle of Enzyme Catalysis • Linus Pauling (1946) formulated the first basic principle of enzyme catalysis – Enzyme increase the rate of a chemical reaction by binding tighter and stabilizing the transition state of its specific substrate more than the ground state – Therefore, higher affinity of the enzyme for the transition state plays an major role in determining substrate specificity
Transcript
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ENZYMES
Serine ProteasesChymotrypsin, Trypsin, Elastase,
Subtisisin
Principle of Enzyme Catalysis
• Linus Pauling (1946) formulated the first basic principle of enzyme catalysis– Enzyme increase the rate of a chemical reaction by
binding tighter and stabilizing the transition state of its specific substrate more than the ground state
– Therefore, higher affinity of the enzyme for the transition state plays an major role in determining substrate specificity
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Enzymes decrease the activation energy of chemical reactions
Enzymes decrease the activation energy of chemical reactions
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Michaelis-Menten Steady-State Kinetic Model
• Leonard Michaelis and Maud Menten (1913)• Catalytic reaction is divided into two processes
– Formation of the enzyme substrate complex via non-covalent interactions (rapid and reversible, “no chemical changes”)
– Conversion of substrate to product (Chemical reaction)
• Michaelis-Menten Steady-State Approximation– The concentration of the enzyme-substrate
complex is constant
Michaelis-Menten Steady-State Kinetic Model
• Michaelis-Menten steady-state approximation is a good approximation if the rate measured are restricted to a short time interval over which the concentration of the substrate does not greatly change
concentration of enzyme is negligible compared to concentration of substrateinitial rate measured
• Pre-steady state: – concentration of intermediates build up to their
steady state• Steady-state:
– reaction rate changes relatively slowly with time– rates of enzymatic reactions are traditionally
measured during this period
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Michaelis-Menten Steady-State Kinetic Model
• The use of pre-steady state kinetics is superior as a means of analyzing the chemical mechanisms of enzyme catalysis
• Steady state kinetics is more important for the understanding of metabolism since it measures the catalytic activity in the steady state conditions of the cell
Michaelis-Menten Equation
[ ]
[ ] [ ][ ] [ ] [ ]
[ ] [ ][ ]
[ ] [ ] [ ]
[ ] [ ][ ]
[ ] [ ][ ]
[ ] [ ][ ]SK
SEkv
SKSE
Sk
kkE
ES
ESEE
kkSEkES
ESkESkSEkdtESd
ESkdtdP
PEESSE
M
Tcat
M
T
cat
T
T
cat
cat
cat
kk cat
+=
+=
++
=
+=
+=
=−−=
=
+⎯⎯→⎯⎯→←+
−
−
−
1
1
1
1
11 0
1
k-1
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Michaelis-Menten Equation
• At high concentration of substrate
• At very low concentration of substrate
[ ][ ] [ ]
[ ] [ ]TcatM
Tcat
M
EkSKSEk
Vv
KS
≈+
==
>>>
max
[ ][ ] [ ]
[ ][ ] [ ] [ ]
MM
Tcat
M
Tcat
M
KSV
KSEk
SKSEk
v
SK
max==+
=
>>>>
•Rate is directly proportional to concentration of enzyme•Rate follows saturation kinetics with respect to concentration of substrate•At sufficiently low [S] rate increases linearly with [S]
Proteinases/ Proteases
• Functions– In viruses: Cleave presursor molecules of the coat
proteins– Bacteria produce many different extracellular
proteinases to degrade proteins in their surrondings– Higher organisms use proteases for
• Food digestion• Cleavage of signal peptides• Control of blood pressure, clotting
– In vivo, the activity of many proteases is controlled by endogenous protein inhibitors
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Proteinases/ Proteases
• Four functional familiesSerine proteasesCysteine proteasesAspartic proteasesMetallo proteases
• Classification based on functional criterion:The nature of the most prominent functional group in active site
Protease Reaction
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Serine Protease Reaction
• All serine proteases use a Catalytic Triad to hydrolyze peptide bonds– Serine– Histidine– Aspartic acid
Serine Protease Reaction
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Serine Protease Reaction
Structural Requirement for Catalytic Action
A general base (His) that can accept a proton from the hydroxyl group of the reactive serine Tight binding and stabilization of the tetrahedral transition state
Oxyanion hole-provision of groups that can form hydrogen bonds to the negatively charged oxygenPositive charge on histidine
Non specific binding of the main chain: Serine proteases have no absolute substrate specificity
Main chain forms a short anti parallel beta sheet with a loop region of the enzymeOne of the H-bond in enzyme-substrate complexes (3.6A)H-bond in complexes mimics of transition state is shorterNonspecific binding contribute to stabilization of the transition state
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Structural Requirement for Catalytic Action
Structural Preference for particular side chain before the scissile bond
Specificity pocket must accommodate the side chain in terms of interactions and size
Specificity Pocket of ChymotrypsinJames at al., J. Mol. Biol. 144:43-88, 1980
• In vivo, chymotrypsinis a proteolyticenzyme acting in the digestive systems of mammals and other organisms.
Inhibitor: Ac-Pro-Ala-Pro-Tyr-CooH
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Specificity Pocket of “trypsin”C.S. Craik et al., Science 228:291-297, 1985
Mutant (Ala216, Ala226)+ Lys Mutant + benzamide
Chymotrypsin: two antiparallel β-barrel domains
Three polypeptide chains
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Chymotrypsin: two antiparallel β-barrel domains
Trypsin
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Bacterial Protease: Subtilisin
• Alpha/beta structure• Added to detergents in
washing powder to facilitate removal of proteinaceous stains
What would happen if Gly226 and Gly216 in trypsin were mutated to Ala?Model building shows that Arg and Lys-containing substrate should be accomodated. Ala226 expected to accommodate Lys better than Arg. With Ala216 the opposite is trueWould kcat, Km and the specificity constant (kcat/Km) change? How? Why?
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Engineered mutants in the substrate specificity pocket change the rate of catalysis
Engineered mutants in the substrate specificity pocket change the rate of catalysis
• Mutants were designed to change specificity• But, the largest change occurred in the catalytic
rates• Interpretation
– Mutations affect the structure of the enzyme in additional ways, possibly causing conformational changes outside the specificity pocket
– These conformational changes reduce the stabilization of the transition state and the activation energy of the reaction
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Lesson/THM
• Critical amino acid residues can have multiple roles in determining a protein’s structure and therefore its function
