Download Serine Proteases

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
1
Enzymes decrease the activation energy of
chemical reactions
Enzymes decrease the activation energy of
chemical reactions
2
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
¾ concentration of enzyme is negligible compared to
concentration of substrate
¾ initial 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
3
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
k1
cat
⎯
E + S ←⎯→
ES ⎯⎯→
E+P
k-1
dP
= k cat [ES ]
dt
k
d [ES ]
= k1 [E ][S ] − k −1 [ES ] − k cat [ES ] = 0
dt
[ES ] =
k1 [E ][S ]
k −1 + k cat
[E ]T = [E ] + [ES ]
[E ]T
[ES ] =
k −1 + k cat
+ [S ]
k1
v=
=
[E ]T [S ]
K M + [S ]
k cat [E ]T [S ]
K M + [S ]
4
Michaelis-Menten Equation
• At high concentration of
substrate
[S ] >>> K M
v = Vmax =
•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]
k cat [E ]T [S ]
≈ k cat [E ]T
K M + [S ]
• At very low concentration
of substrate
K M >>>> [S ]
k [E ] [S ] k cat [E ]T [S ] Vmax [S ]
=
=
v = cat T
K M + [S ]
KM
KM
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
5
Proteinases/ Proteases
• Four functional families
¾ Serine proteases
¾ Cysteine proteases
¾ Aspartic proteases
¾ Metallo proteases
• Classification based on functional criterion:
¾ The nature of the most prominent functional group in
active site
Protease Reaction
6
Serine Protease Reaction
• All serine proteases use a Catalytic Triad
to hydrolyze peptide bonds
– Serine
– Histidine
– Aspartic acid
Serine Protease Reaction
7
Serine Protease Reaction
Structural Requirement for Catalytic Action
¾
A general base (His) that can accept a proton from the
hydroxyl group of the reactive serine
¾ Tight binding and stabilization of the tetrahedral
transition state
¾ Oxyanion hole-provision of groups that can form hydrogen
bonds to the negatively charged oxygen
¾
¾ Positive charge on histidine
Non specific binding of the main chain: Serine
proteases have no absolute substrate specificity
¾
¾
¾
¾
Main chain forms a short anti parallel beta sheet with a loop
region of the enzyme
One of the H-bond in enzyme-substrate complexes (3.6A)
H-bond in complexes mimics of transition state is shorter
Nonspecific binding contribute to stabilization of the transition
state
8
Structural Requirement for Catalytic Action
¾ Structural Preference for particular side chain
before the scissile bond
¾ Specificity pocket must accommodate the side
chain in terms of interactions and size
Specificity Pocket of Chymotrypsin
James at al., J. Mol. Biol. 144:43-88, 1980
• In vivo, chymotrypsin
is a proteolytic
enzyme acting in the
digestive systems of
mammals and other
organisms.
Inhibitor: Ac-Pro-Ala-Pro-Tyr-CooH
9
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
10
Chymotrypsin: two antiparallel β-barrel
domains
Trypsin
11
Bacterial Protease: Subtilisin
• Alpha/beta structure
• Added to detergents in
washing powder to
facilitate removal of
proteinaceous stains
• Catalytic triad
• Red:polypeptide inhibitor
• Purple: oxyanion hole
• Orange; non-specific
binding
a protease secreted by a soil bacillus
Active Site of Subtilisin
• Red: bound
polypeptide inhibitor
(eglin)
• Catalytic triad:
Ser221, His64, Asp32
• Oxyanion hole:
Asn155
• Specificity pocket
• Non-specific binding
of peptide
12
Chymotrypsin
Subtilisin
Probing the Role of the Specificity Pocket
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 true
Would kcat, Km and the specificity constant (kcat/Km) change?
How? Why?
13
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
14
Lesson/THM
• Critical amino acid residues can have multiple
roles in determining a protein’s structure and
therefore its function
15