Enzyme Structure/Function and Kinetics Flashcards
What is a Zymogen?
How is it related to the blood coagulation cascade?
Zymogens are inactive enzymes that is inhibited by part of its own AA sequence. It is cleaved or taken away by the previous active enzyme in the cascade.
These zymogens have little to no enzymatic activity until they are activated to become serine proteases.
example: thrombin hydrolyzes fibrinogen into fibrin.
Usually, the inactive zymogen is -ogen, while the active enzyme is without that ending.
What does Thrombin do?
Thrombin hydrolyzes the peptide bond in fibriongen to make it into fibrin.
it hydrolyzes the peptie bond in between the Pro-Arg and Gly sequence.
H-bonding allows the Ser-His-Asp to be near each other in the tertiary structure, but in the primary structure.
What is the rate limiting step and how do we know it is?
The rate-limited step is the breaking apart of the covalent bonds of the acyl-intermediate from the active site of the enzyme.
We know this because of the pre-steady state burst, which proves there is a somewhat stable acyl-intermediate in the reaction mechanism.
We are in steady state when [P] > [E].
What is the Catalytic Triad for Serine Proteases
S-H-D
Ser-His-Asp
Serine-Histidine-Aspartic Acid
What are te Binding Residues for…
a) Trypsin
b) Chymotrypsin
c) Thrombin
a) R1: Lys (K) and Arg (R) that interact with partial negative charge of serine and aspartic acid
b) R1: Phe (F), Trp (W), and Tyr (Y) fit into pockets
c) R1: Arg (R) interacts with partial negativ charge of serine.
R2: Proline: fits in pocket
What is Ki?
Dissociation constant of the inhibitor to the enzyme.
Therefore large=bad=high V
small=good=low V
It is the concentration of the inhibitor needed to bind to 50% of the enzyme molecules.
What is the difference beteween the two types drug design?
Rational Drug Design: based on mechanism of reaction
Complimentary Shape Design: based on structure
Serine Protease Mechanism
1) The hydroxy-group on serine donates electrons to slightly positice carboxyl carbon between R and G, caausing a transition state
2) The transition state is decomposes, causing G to leave (P1) and leaving the EI still intact
3) Hydrolysis of EI at carboxyl carbon makes another transition state, causing the S to leave
4) LEft with P2 (P-R) and Enzyme (Ser)
What is the energy profile for a reaction with
a) No enzyme
b) Enzyme complimentary to substrate
a) substate requires a lot of energy to reach bent transition state. Energy is released upon breaking.
b) substrate binds to enzyme but cannot bend to transition state
Energy Profile when enzyme is complimentary to Transition State?
1) Covalent bonds break (increase energy) and new bonds are formed as substrate binds to enzyme (lowered energy to form ES complex.
Destablize the ES complex (1st valley) via strain, desolvation, loss of entropy, conformation change of enzyme.
2) Since the enzyme compliments the shape of the transition state via hydrogen bonds, hydrophobic interactions, charge-charge interactions to make tetrahedal shape, substrate can bend into transition state and be stabilized and lower what would be the transition state energy (increase in energy)
3) The substrate breaks apart into P1 and EI (decrease in energy).
4) Hydrolysis is needed to break EI (increase in energy(, followed by breaking off of P2 and E (decrease in energy).
General Base Catalysis.
What acts as the base? Why is it needed?
1) HIS-57 is needed as a general base do donate electrons to the Oxygeon in Ser-195 to make it more basic in order for it to react.
ASP-102 donates electrons to histidine-57 as well to make it donate its electons to Ser-195.
2) Hydrogen bonds from the enzyme act on the Oxygen in the transition state to stabilize the tetrahedral shape/negative charge.
3) Then Acid Catalysis happens
General Acid Catalysis
The same general base His-57 is the acid in this case.
1) Accepts electrons from N from transiton state to promote breaking of peptide bond
Transition state breaks down and gives of P1 (Gly) and leaves EI complex which is the acyl-intermediate
2) Hydrolysis of active site at carboxyl carbon of EI to make another transition state and N of HIs-57 acts as a base. Rate-limiting step
3) Breaking of acyl-enzyme intermediate bond with Ser-195 as transition state breaks down to make E + P2 (Pro-Arg) complex
4) P2 is released from enzyme
Transition State Analagues
An enzyme stabilizes the transition state. So, you want to design a drug that mimics the shape and charge of the transition state.
You also want a low Ki.
