BIOL 302 1st Edition Lecture 12Outline of Last Lecture I. Test 1 Study GuideOutline of Current Lecture II. Two Substrate ReactionsIII. Proximity and orientation effectsIV. Preferential binding of the transition state complexV. Catalysis by destabilizing the substrateVI. Acid-base catalysis, Covalent catalysis (electrophilic and nucleophilic catalysis)VII. Metal ion catalysisCurrent LectureCurrent LectureI. Two substrate reactionsA. Ordered Sequential1. Ordered = all reactants in before reaction occursB. RandomThese 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.C. Ping Pong1. Substrates and products alternate in and outII. Enzymes are catalysts and reduce the activation energyIII. Stabilization of the transition stateA. Think of substrate S and transition state X‡ to be in equilibrium B. Rate of reaction is directly proportional to X‡C. The smaller the difference in free energy between S and X‡, the greater the concentration of X‡IV. Tight binding of S‡ to E is catalyticA. An enzyme with a pocket complementary tothe reaction transitionstate helps to destabilize the substrate, therebycontributing to catalysis.V. Catalytic mechanisms of enzymesA. Proximity and orientation effectsB. Preferential binding of the transition state complexC. Catalysis by destabilizing the substrateD. Acid-base catalysisE. Covalent catalysis (electrophilic and nucleophilic catalysis)F. Metal ion catalysisVI. Catalytic mechanisms of enzymesA. Catalysis can occur though proximity and orientation effects. B. Enzymes catalyze reactions by preferentially binding the transition state or by destabilizing the substrate.C. Acid-base catalysis, uses e.g. side chains of Asp, Glu, His, Cys, Tyr and Lys, which have pK’s in the physiological range.D. Covalent catalysis, which requires usually nucleophiles, such as the imidazole group of His, the unprotonated amino group ofLys, the thiol group of Cys, the carboxyl group of Asp and the hydroxyl group of Ser, or coenzymes such as thiamine pyrophosphate. E. Catalysis by metal ion cofactors, most commonly tightly bound transition metal ions such as Fe2+,Fe3+, Cu2+, Mn2+, Co2+, Mg2+ and Zn2+.VII. How to read reaction mechanismsA. The arrow always goes from electron-rich to electron-poor groupVIII. 1. Proximity and orientation effects – Complex IIIIX. Complex III from the respiratory chain is an enzymeA. QH2 + 2 cytochrome c(Fe3+) + 2H+N→Q + 2cytochrome c(Fe2+) + 4H+P .X. Proximity and orientation effects – Complex IIIA. Binding of cytochrome c to subunit cytochrome c1 of Complex III and preoriientation of these protein partners is required for heme-to-heme electron transfer.XI. Catalysis by preferentially binding the transition stateXII. Catalysis by preferentially binding the transition state– catalytic triad of serin proteasesXIII. Catalysis by preferentially binding the transition state– catalytic triad of serin proteasesXIV. Catalysis by preferentially binding the transition state– catalytic triad of serin proteasesA. After the protease has bound a protein substrate, Ser195 nucleophilically attacks the scissile peptide’s carbonyl group to form the tetrahedral intermediate.XV. Catalysis by preferentially binding the transition state – catalytic triad of serin proteasesA. Left: the trigonal carbonyl carbon atom of the scissile peptide cannot bind in the oxyanion hole.B. Right: In the tetrahedral intermediate, the oxyanion (carbonyl oxygen of the scissile peptide) hydrogen bonds to the NH groups of Gly193 and Ser195. This conformation alsopermits the NH group of the preceding amino acid to form a hydrogen bond to the CO group of Gly193. => Stronger BindingXVI. Evidence for existence of the tetrahedralintermediate – x-ray structure of trypsin and its inhibitor BPTI A. BPTI (green): The inhibitor arrests the normal proteolysis reaction closely preceding the tetrahedral intermediate.XVII. Catalysis by substrate destabilizationA. Tight binding of S to E in the ground state is anti-catalyticB. When the enzyme is complementary to the substrate, the ES complex is more stable andhas less free energy in the ground state than substrate alone. The result is an increase in the activation energyXVIII. Catalysis by substrate destabilizationA. Destabilization can facilitate catalysis provided that (1) the energy of S is brought closer to X‡ and (2) destabilization is relieved at transition stateXIX. Catalysis by substrate destabilizationA. Destabilization raises the free energy of the substrate, bringing it closer to the transition state.XX. Catalysis by substrate destabilizationA. Destabilization through entropy reduction: decreasing the degrees of freedom available to the substrate increases its free energy, making it more reactive.XXXIII. Catalysis by substrate destabilizationA. Destabilization by desolvation. Removing a very polar functional group from water raises its energy, making it more reactive.XXI. Catalysis by substrate destabilizationA. Destabilization by “stress”, electrostatic or steric. Juxtaposition of two like charges raisesthe free energy of the substrate, making it more reactive.XXII. Acid base catalysisA. "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center B. "General" acid-base catalysis involves acids other than H+ and bases other than OH- C. These other acids and bases facilitate transfer of H+ in the transition stateXXIII. Acid base catalysis – RNase A – first stepA. General acid catalysis: proton transfer from an acid lowers ∆G of the transition state. General base catalysis: a proton is abstracted by a base. B. In RNase A, His12, acting as a base, abstracts a proton from an RNA 2’ OH group, therebypromoting its nucleophilic attack on the adjacent P atom. His119, acting as a general acid, promotes bond scission by protonating the leaving ribonucleotide.XXIV. Acid base catalysis – RNase A – second stepA. Water enters the active site, and the cyclic intermediate is hydrolyzed through the reverse of the first step. B. Thus, His12 now acts as a general acid and His119 as a general base to yield the hydrolyzed RNA and return the enzyme to its original state.XXV. Covalent catalysisA. Covalent catalysis accelerates the reaction through the transient formation of a catalyst-substrate covalent bond. B. This bond is usually formed by the reaction of a nucleophilic group on