Chapter 6 Lehninger Principles of Biochemistry PDF

6| Enzymes © 2017 W. H. Freeman and Company  Rate vs Direction of Biochemical Reactions Direction is predicted by the change in free energy of the overall reaction, ∆Grxn – Free energy is the energy that becomes available to do work as a reaction approaches equilibrium – ∆G...

6| Enzymes © 2017 W. H. Freeman and Company  Rate vs Direction of Biochemical Reactions Direction is predicted by the change in free energy of the overall reaction, ∆Grxn – Free energy is the energy that becomes available to do work as a reaction approaches equilibrium – ∆Grxn = ∆Hrxn- T∆Srxn – ∆Grxn < 0 for spontaneous reactions ATP is said to be thermodynamically unstable but kinetically stable. Explain using a free energy diagram for ATP hydrolysis. ∆Go = -30.5 kJ/mol. – Note the difference between ∆Grxn and ∆G++ (Ea)  Biochemical Kinetics What factors influence rates of reactions? – What would be the rate law for A + 2B  C assuming this reaction occurs by a single elementary step and is therefore the slow or rate-determining step? Rate = k [A][B]2 Concentrations of reactants Rate constant, k, is defined by the Arrhenius equation and includes the temperature and energy of activation (Ea = ∆G++ )  Enzymes lower the activation energy to increase the reaction rates for both the forward and reverse directions Note that catalysts do NOT change the ∆G (direction, equilibrium position) of a reaction  Catalysts Increase rate towards equilibrium by lowering ΔG++ Enzymes can provide 106-1014 or greater rate enhancements! Table 11-1 Table 11-2 Classes of Enzymes  Enzyme Nomenclature End in -ase Common names – Often ambiguous What does catalase do? Systematic names – Established by enzyme commission of IUBMB Example – Common: carboxypeptidase A – Systematic: peptidyl-L-amino acid hydrolase EC.3.4.17.1 Figure 11-1  Enzymes are Stereospecific and Can Distinguish Groups on Chiral or Prochiral Centers Figure 11-2 Enzymes Often Require Help Cofactors required for transfer reactions – Metal ions – Organic molecules (coenzymes) Transiently bound (cosubstrates) Tightly bound (prosthetic groups) NAD+/NADP+ are cosubstrates  How do enzymes lower ∆Gⱡ? Binding and orientation of substrate(s) to increase reactivity and proximity of chemical groups Preferential binding of Xⱡ Provide an alternative pathway w/ lower ΔGⱡ  Chemical Bases for Enzymatic Catalysis Acid-base catalysis (H+ transfer) – RNase overhead Covalent catalysis (transient covalent bond) – Decarboxylation of acetoacetate overhead – Nucleophilic groups overhead Metal ion catalysis – Metalloenzymes Transition metal ions in active site that play role in catalysis – Fe, Cu, Zn, Mn, Co Contributions to electrostatic catalysis (see below) Increase reactivity of H2O by polarization Redox reactions Electrostatic catalysis – Hydrogen bonding/ionic bonding involving amino acids or metal ions – Preferential stabilization of transition state – Binding and orientation – Guidance of S to active site Acid-Base Catalysis Figure 11-8  Amino Acids in General Acid-Base Catalysis  Specific Example: RNase Two key histidine residues in active site – His 12 = base – His 119 = acid An example of a hydrolysis reaction – A common type of reaction – Key question: Is H2O present initially in the active site? Figure 11-10 part 1 Figure 11-10 part 2 Covalent Catalysis Figure 11-11  Formation of a Schiff Base Page 330 Figure 11-12 Roles of Metal Ions in Catalysis Electrostatic effects – Stabilization of X++ – Binding and orientation of S Electron transfer (redox) Polarization of H2O – E.g., the carbonic anhydrase reaction Charge-shielding on substrate – See Mg-ATP  The Serine Proteases One class of proteolytic enzymes Catalyze hydrolysis of peptide bonds – Chymotrypsin Carboxyl end of bulky, hydrophobic (Phe, Trp, Tyr) – Trypsin Carboxyl end of positively-charged (Arg, Lys) – Elastase Carboxyl end of small, neutral amino acid residues Substrate Specificity – Specificity binding pocket Enzyme Catalysis – Catalytic triad promotes alternative pathway Ser, His, Asp – Oxyanion hole preferentially stabilizes X++ by H-bonding Figure 11-27 Figure 11-29 Figure 11-30a Figure 11-30b Figure 11-15  Design a stable transition state analog that you would predict to be a potent inhibitor of proline racemase Page 339 Page 339 Protein Tyrosine Phosphatases CX5R active site H2O not present initially Mechanism? Serine/Threonine Phosphatases Water present initially? Role of metal ions? Role of His 125? ENZYME KINETICS  Why Study Enzyme Kinetics? Quantitative description of biocatalysis Determine the order of binding of substrates Understand catalytic mechanism Find effective enzyme inhibitors Understand regulation of activity  Introduction to Enzyme Kinetics Consider simple two-step, one-substrate model Assumptions ES  E + P is the slow step Measuring initial rate, v0 ES is in a “steady state” Figure 12-2  Michaelis-Menten Equation THE rate equation for non-allosteric enzymes Write a rate equation from the rate-limiting step vo = k2[ES] Must substitute [ES] w/ terms that are measurable Assume steady state: rateformation = ratebreakdown k1[E][S] = k-1[ES] + k2[ES] Let [E] = [ET]-[ES] and rearrange to collect rate constants (k-1 + k2)/k1 = KM = {([ET]-[ES])[S]}/[ES] – the Michaelis constant  Michaelis-Menten Eq. (cont.) Solve for [ES] [ES] = [ET][S]/(KM + [S]) Recall that v0 = k2[ES], so [ES] = v0/k2 Substitute [ES] with v0/k2 and solve for v0 v0 = k2[ET][S]/(KM + S) Since vmax= k2[ET], substitute k2[ET] with vmax V0 = Vmax[S]/(KM + [S])  Significance of KM [s] required for the enzyme to do it’s thing – KM = (k-1 +k2)/k1 = s-1/M-1s-1 = mol/L – From MM eq, [S] = KM when V0 = ½ Vmax Related to the equlibrium binding affinity of E for S – [ES]  [E] + [S] – at equilibrium, k-1[ES] = k1[E][S] – KD = [E][S]/[ES] = k-1/k1 – KM = (k-1 +k2)/k1 – KM ~ KD when k2 2 steps kcat is a more general term for k2 1st order rate constant for the slow step of any M-M E Since vmax = k2[ET], we can write vmax = kcat[ET] Solving for kcat = vmax/[ET] = Ms-1/M = s-1 # SP/enzyme molecule/s = “molecular activity” or “turnover #”  Specificity Constant, kcat/KM A measure of the catalytic efficiency Recall v0 = vmax[s]/(kM + [s]) and vmax = kcat[ET] So vo = kcat[ET][s]/(kM + [s]) Efficiency is considered at low [s] where [s]