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CHEM 4465

ENZYMES (DR. NUGENT) I. INTRODUCTION TO ENZYMES/CATALYSTS a. Definition: enzyme = biological catalyst (catalyst = speeds rate of reaction) b. Features of enzymes: i. Enzymes speeds up the rate of a reaction 1000000 fold, up to 10^12  reaction can now fulfill a need ii. enzymes are specific – to certain substrates or to class of compounds (this is different from a general catalyst such as Ni or Pt) iii. enzymes put compounds in an environment where certain reactions are now accelerated (don’t want to change environment of entire system, ie body ph, so need active site of enzyme to create micro environment) iv. enzymes CANNOT: 1. change equilibrium of a reaction 2. be consumed or changed (they are always regenerated) II. THERMODYNAMICS OF CHEMICAL REACTIONS a. Terms and Equations i. G = free energy; G°= G products G reactantsf G° < 0 then reaction is spontaneous ii. Keq Product/Reactant; if K > eqthen reaction is spontaneous (there are more prod than reactants at equilibrium) iii. G° = RTlnK ;eq = constant and T = temp b. Interpretation of G: i. High free energy means thermodynamically unstable *relative terms { ii. Low free energy means thermodynamically stable(high probability that compound will stay in this state) iii. G° for hydrolysis of glucose = -686 kcal/mol  means glucose is very unstable BUT kinetically stable (refers to time frame)… c. Activation energy ‡ i. Activation energy (G ) is the barrier the reaction must get past ii. transition state = least stable conformation of reactant (state in which all C-C bonds are half-broken, half-formed)  very unlikely reactant will stay in this state : iii. The rate of the reaction is defined by the activation energy ‡ ‡ 1. G = G transition staground G > 0 2. A catalyst lowers activation energy (G ) ‡ ‡ iv. Ways enzymes reduce G : 1. By altering the course of a reaction: many enzymes work together in a pathway (“staircase analogy”) 0 2. By destabilizing the ground state higher G ; smaller G 3. By stabilizing the transition state  lower G trans smaller  G III. RATE THEORY a. Equations i. G = -RTlnK = G‡ – G ‡ trans ground ii. K = [T]/[ground] <<1 (small fraction because T is very unstable) iii. AP is an irreversible reaction, and velocity is not constant (A gets used up) so to find velocity: d[P]/dt = V = k[A] M/sec k = rate constant *this is a first order reaction ‡ b. Derivation of relationship of G to k v=6.2x10^12/sec i. d[P]/dt = k[A] Bolt’s constant, the theoretical frequency of ii. k [A] = v[A ] (rate constant relates to activation energy) how fast things change if ‡ T ‡ there is no barrier iii. G = -RTlnK = -RTln[A ]/[A] ‡ iv. G = 17.4 – 1.36 log k ‡ 1. So G relates to log of the rate constant k G k 2. if G decreases, then k increases exponentially: 17.4 1 c. Reverse reaction 13. 4 1000 i. d[P]/dt = k[A] – k [P] = 0 at equilibrium f r ii. K eq[P]/[A] = k/k f r iii. Tells us relative speeds: if K >eq, then k > k f r iv. *enzymes cannot change preference: a catalyst that speeds up k speeds up k tf r an equal magnitude (eg enzyme that accelerates phosphorlyation also accelerates dephosphorylation IV. ENZYME CATALYSIS IN PHYSIOLOGY AND MEDICINE a. The Blood Clotting Cascade - Introduction i. The circulatory system is a closed system. A cut violates this closed system  body needs to stop bleeding without changing circulation everywhere else  need to change liquid to solid (clot) in one area ii. Biochemistry has certain time constraints  need enzymes to speed a reaction exponentially (1 enzyme  1,000,000 enzymes  1,000,000,000,000 iii. Enzymes can not only speed up a reaction; they can also speed up the reactions of other enzymes. iv. Proteases change circulating proteins; some change activity of other enzymes b. Blood Clotting Cascade – Mechanism i. Fibrinogen – a protein in the blood that is soluble (has hydrophilic residues) ii. Thrombin (IIa) – a protease 1. clips off “hairs” on fibrinogen 2. hydrophilic region is exposed 3. fibrin self-associates to form fibrin clot (precipitates) iii. Thrombin is not floating in blood; it is made by activating prothrombin (made in liver) 2 iv. Factor Xa (made from X, which is in blood) clips prothrombin to make thrombin v. Factor IXa activates factor X vi. Factor VIII activates IX So: there are multiple ways to activate enzymes, and the clotting can be controlled at various points in cascade. c. Control of Blood Clotting i. Vitamin K 1. injected in newborns to prevent hemorrhagic disease 2. collaborates with  glutamyl carboxylate in liver to activate prothrombin (also factor X, others) 3. takes days-weeks to build up clotting capabilities with vitamin K ii. Blood clotting inhibitors: 1. Warfarin a. acts at beginning of cascade b. Acts in liver: antagonizes vitamin K c. prevents prothrombin (II) and factor X from being sufficiently carboxylated - can’t be active d. inhibits clotting e. Examples = d-Con, Coumadin 2. Antithrombin III = natural inhibitor a. acts at end of cascade b. inactivates thrombin(IIa) – forms a covalent cross-link and kills it c. antithrombin III binds to a healthy surface on endothelium, changes into more active conformation d. heparin sulfate (linear polysaccharide) = compound expressed by endothelium e. *Heparin can be isolated and used as an anticoagulant 3. Mechanism for eliminating a clot: a. T-PA (tissue plasminogen activator) - enzyme that activates plasminogen into plasmin (a protease) b. Plasmin “chews up” fibrin (insoluble) into soluble products* *not reusable – blood clotting is only for “emergencies” because it is “expensive”; entire system requires resynthesis c. can’t administer plasmin (would need too much!) but can administer t-PA, which then activates exponentially more plasmin. d. Returns system back to normal Q: How would you treat a Coumadin overdose? A: With vitamin K 3 V. ENZYME MECHANISMS Enzymes can reduce G in a number of ways: a. Alter course of reaction – break an unlikely event into multiple steps i. G is log related to K – drop G by a little  increase K exponentially b. Destabilization of substrate/Stabilization of transition state i. Geometric – twisting, turning, pulling (ie tetrahedral conformation into planar – induces formation of double bonds) ii. Electrostatic – put compound in a different environment  raise free energy iii. Desolvation – make compound no longer soluble (ie by excluding water in active site) c. Two models of enzyme specificity i. Lock and Key (older hypothesis) 1. Enzymes are geometrically (or chemically) oriented so they have an active site that is complementary - a mirror image - of substrate binding site 2. Meeting of two chemical groups  reaction is favored 3. Suggests that enzymes evolved to be perfect fits for substrates 4. Formal definition of substrate binding site= region of enzyme that makes noncovalent contact with substrate such that it can hold the substrate in place 5. Active site = components on the enzyme that are actively involved in chemical catalysis (doesn’t have to be same as substrate binding site) ii. Induced fit (newer – addresses destabilization) 1. Evolutionary pressure is for enzyme to be complementary to the transition state so “if it ever found transition state around it would bind very nicely” 2. BUT enzyme never finds transition state (unlikely conformation because unstable)  enzyme tries to find substrate that somewhat resembles, and then changes 3. Both substrate and enzyme are strained a little to destabilize ground state, make transition state a little more likely  more likely to react VI. ENZYME KINETICS - Michaelis-Menten Equation a. Reaction Model i. E + S ES E+P ii. Initial Rate approximates linear generation (given rate of reaction) iii. Assumptions: [S] = 1 1. No reverse reaction occurring 2. Neglect Product Inhibition (Feedback inhibition)
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