BIOL 302 1st Edition Lecture 14Catalytic StrategiesOutline of Last Lecture I. Chymotrypsin Reaction MechanismII. ProteasesOutline of Current Lecture II. ZymogensIII. FibrinIV. Carbonic AnhydraseV. Restriction EnzymesVI. Myosin and ATP HydrolysisCurrent LectureI. ZymogenA. activation by limited proteolysisII. Zymogen ActivationA. proteolytic cleavage of trypsinogen leads to protease cascadeThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.III. CascadesA. Of proteolytic events activate prothrombin to thrombin, so as to proteolytically cleave fibrinogen.IV. Polymerization of Fibrinproduces clotV. Polymerization of Fibrin produces clotVI. Carbonic AnhydraseA.Electrophilic CatalysisB.Catalysis by metal ionsC.pH effectsVII. Carbonic AnhydraseVIII. Carbonic AnhydraseA. A Zinc2+ ion is always bound to four or more ligands.B. Note that these histidines are almost neutrally chargedIX. Carbonic AnhydraseA. pH effect on the kcat for carbonic anhydraseB. Zn++ allows water to lose a proton at neutral pH, makes it into a strongnucleophile to attack CO2X. Carbonic AnhydraseA. CO2 binding siteB. Carbonic anhydrase has a hydrophobic binding pocket for CO2 and brings it close to the Zinc-bond waterXI. Carbonic AnhydraseA.The Zinc ion facilitates release of a proton from waterXII. Carbonic AnhydraseA. CO2 binds to the active site and is positioned to react with the hydroxide ion.XIII. Carbonic AnhydraseA. The hydroxide ion acts as nucleophile to attack + C of CO2, converting it into the bicarbonate ion HCO3-XIV. Carbonic AnhydraseA. The catalytic site is regenerated, with release of the bicarbonate ion and the binding of another water molecule.XV. Carbonic AnhydraseXVI. Carbonic AnhydraseA. proton shuttle1. His 64 helps to shuttle the proton from water to the surface of the protein, to be picked up by buffer.2. Without buffer, kcat becomes limited by proton diffusionXVII. Clicker question: What are the roles of his96, his119 and his94 in the catalytic mechanism of Carbonic anhydrase? A. Binding site for water. B. Binding site for zinc ion. C. Proton shuttle. D. General acid and general base.E. All of the above.XVIII. Restriction EnzymesA. There are three types of restriction enzymes: I, II & III. B. Type II enzymes are used for cloning and sequencing DNA.C. Hydrolysis by Type II enzymes generates a 3’ OH and 5’ phosphate.1. Electrophilic Catalysis2. Catalysis by metal ions3. Substrate distortionXIX. Mg++ needed by the Type II enzymeXX. Restriction endonucleases distort the correct (cognate) substrateA. Enzyme complexed with Cognate and Noncognate DNAB. Distortion drives binding of Mg2+ at the active siteXXI. Extra Binding Energy with Cognate Substrate Drives DistortionXXII. Myosin and ATP hydrolysisA. Myosin is the most comprehensively studied molecular motor B. This enzyme converts energy from the hydrolysis of MgATP into directed movement1. Conformational Change2. Solution vs. “Internal” EquilibriumXXIII. ATP – energy currency of the cellA. A large amount of free energy is liberated when ATP is hydrolyzed (∆G○’= -7.3 kcal/mol)B. Why?1. Resonance increased 2. Repulsion relieved3. Hydration enhancedXXIV. ATP hydrolysis proceeds by direct water attackA. ATP:Mg+2 is the true substrate for this enzymeB. Reaction is assisted concerted general acid/base catalysisXXV. Change in myosin conformation must accompany myosin activityA. Soaking crystals of myosin in a solution containing ATP:Mg+2 did not lead to hydrolysisXXVI. Use of a transition state analogue to lock myosin in its catalytically competent stateA. Vanadate ion can adopt a trigonalbipyramidal coordination that has been proposed to mimic the conformation of the phosphate group at the transition state expected for ATP hydrolysisB. The half-life for dissociation of the myosin-ADP-Vi complex is 4 days !!XXVII. The free energy of ATP hydrolysis can be put to work by myosinA. Comparison of substrate and transition state analogue complexes reveal remarkable change in myosin conformation1. Active site2. Carboxyl terminusXXVIII. Isotope exchange and the internal equilibriumA. ATP hydrolysis is “irreversible” in water, but at the active site of myosin the reaction is freely reversible .B. Each ATP is cleaved and reformed several times before the products are released.XXIX. Clicker question: Myosins couple ATP-hydrolysis to conformational changes which facilitate transport of cargo along …………. tracks.A. intermediate filamentB. microtubuleC. actinD. membraneE. all of the aboveXXX. Clicker question: In Myosins, what is the transition state for ATP hydrolysis?A. ADP and vanadate bound at the catalytic site.B. A water molecule covalently bound to magnesium.C. A magnesium ion that bridges ADP and phosphate in the catalytic site. D. A pentacoordinate transition state of phosphate and water.E. All of the aboveXXXI. The P-loop of NTPase domainsXXXII. Clicker question: Which of the following proteins have a mechanism of ATP hydrolysis that is similar to the Myosins? A. KinesinsB. Elongation factor TUC. G-proteinsD. ATP synthaseE. all of the