Main Ideas - Enzymes are catalysts that lower the activation energy for a rxn and speed up the rxn rate!- Binding of a protein to a ligand (1st step of catalysis) can be measured by the binding (equilibrium) constant!- The Michaelis-Menten (MM) equation relates the rate of an enzyme catalyzed rxn to the substrate concentration and two constants, Km and kcat!- Based on the MM equation, the best enzymes have a small Km and a large Vmax (w/ Km= [S] at Vmax/2)!- Ex. of enzymatic amplification thru regulated proteolysis= blood coagulation cascade Learning Objectives 1) Understand the basic principles and steps of enzyme catalyzed rxns - Enzymes are catalysts and are everywhere!- Catalyzed rxns are typically connected in series!- Enzymes lower the activation energy that blocks chemical rxns --> hugely increase the rate/speed of chemical rxns, but don't affect/change Keq (the state where products and reactants are in equilibrium)!- They are highly selective= 1 enzyme: 1 rxn!- Due to active site: pocket only accepts 1 substrate!- Substrate/ligand= molecule that binds to the enzyme!- Binds thru non-covalent interactions and complementary binding surfaces!- Hypothesis: 1) lock and key= fit each other perfectly 2) induced fit= don't fit perfectly initially, enzyme has to undergo a little change to fit perfectly!- Ex. serine protease= attacks peptide bonds on proteins, has a basic microenvi, so due to the interactions between its side chains via H-bonds, a nonreactive group-->reactive group!- Active site: catalytic triad!- First step: substrate binding - Dynamic process: the substrate binds to other molecules, but their interactions are too weak, so they fall apart. Substrate only stays w/enzyme when it's supposed to be bound to it.!- Equilibrium constant measures binding strength!- at equilbrium the association rate = the dissocation rate!- k1= kon!- k2= kcat (catalysis)= ES--> E + P!- this is the limiting step!- ES is a transient intermediate !- understand steady state approximation: ES concentration stays the same, so d[ES]/dt= 0, helps us get to MM2) Understand that the MM eqn describes how rxn velocity depends on substrate concentration - So the velocity of your rxn is the increase in product made over time and this depends on your substrate concentration - vmax!- once all the enzyme is bound by substrate, the max rxn rate is reached!Direct Plot: - When your rxn has reached half of its max v, Km= S - The best enzymes have the smallest Km (least tendency to dissociate) and largest Vmax! 3) Be able to interpret a direct plot or a double reciprocal plot for an enzyme that follows MM kinetics - See above 4) Be familiar w/the basic principle of an enzyme amplification cascade (ex. blood coagulation) - Enzymes and rate acceleration - Range: 10^9-10^23 increase in rate!- Factors that can influence rate:!- Concentration of enzyme and substrate, affinity between E and S, ability of E to stabilize transition state (ES), bond strain, T of rxn and pH of rxn!- Enzyme Cascade: blood coagulation - Hemostatis: arrest of blood loss from ruptured blood vessels!- Primary: vascular restriction + platelets adhere to injury site!- Secondary: coagulation= the casese of rxns that produces a fibrin mesh (clot) that entraps the soft plug from above!- Triggering coagulation!- Membrane proteins exposed by injury serve as receptors for zymogens and protein co-factors that are either: already present in the blood or released by platelets following their activation!- Both intrinsic and extrinsinc cascade using proteolysis to amplify the signal!- Multimembered cascade!- One member gets activated and then activates the next zymogen --> etc.!- One enzyme at the start of the cascade can activate many molecules of a given substrate!- Factors XII, XI, IX, VII, X, and II: zymogens (=precursor) of serine proteases - Cleavage of 1 zymogen-->active protease that activates another zymogen!- Each zymogen is also reg. by protein cofactors that themselves are also reg. by proteases (+ feedback looping-->more amplification)!- zymogens are cleaved (all these lose their N terminal parts to free up the serine protease part) to become
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