BIOL 302 1st Edition Lecture 10 Outline of Last Lecture I. Review II. O2 in Hemoglobin and MyoglobinIII. EnzymesOutline of Current Lecture IV. EnzymesV. Enzyme KineticsVI. Michaelis-Menton plotVII. Lineweaver Burke PlotVIII. Review for first testCurrent LectureIX. Many enzyme active sites have a special affinity for the transition state substrate (X‡)A. If the transition state were not bound more tightly than the ground state, catalysis would not occur.These 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.X. What is the evidence for the existence of a Enzyme-Substrate complex?A. ES is a noncovalent complexXI. What is the evidence for the existence of a Enzyme-Substrate complex?A. (noncovalent)XII. Properties of enzyme active sitesA. Need not be adjacent in B. Primary sequence.C. 3o structure must have them all positioned around the active site.XIII. Properties of enzyme active sitesA. Substrates are bound by enzymes using multiple weak interactions.B. The full complement of these interactions forms at the transition stateXIV. Properties of enzyme active sitesA. Unique microenvironmentB. Engulfs the substrateXV. Effects of pH are important for enzymatic activityXVI. Effects of salt concentration are important for enzymatic activityXVII. How do we identify a catalytic site?A. a crystal structure of the enzyme with its substrate, ligand or cofactor bound, or an inhibitor that mimics the substrate or a transition state.B. If only a crystal structure of the protein by itself is available: 1. Literature search: which residues were identified by mutagenesis experiments as relevant for the catalytic activity? 2. What are their properties?3. Look for homologous proteins and identify structural motifs.4. Identify highly conserved residues.XVIII. Complex III of the respiratory chain: Qo ubiquinole oxidation siteXIX. Lock & Key – Fischer (1894) A. A proposal for ES XX. Enzymes are stereospecificXXI. Induced Fit – Koshland (1963)A. A proposal for ESXXII. Enzyme KineticsA. Branch of enzymology that investigates factors affecting the rates of enzyme-catalyzed reactions. The most important of these are enzyme concentration, substrate/product concentration, pH, ionic strength, temperature, and the presence of inhibitorsB. Analysis of enzyme kinetics can help deduce catalytic mechanism, establish amino acid involved in the transformation, and ascertain the mode of action of enzyme inhibitors.XXIII. Enzyme KineticsA. k1 = second order rate constant for formation of ES from E + S B. k-1 = first order rate constant for decomposition of ES to E + SC. kcat = first order rate constant. Maximum number of substrate molecules turned into product per active-site, per unit timeD. Km = concentration of S where v is ½ vmax. An apparent equilibrium constant for ES dissociationE. kcat/Km = specificity constant, measure of an enzymes catalytic efficiency toward a substrateF. vmax = limiting value of reaction velocity approached [S] ≥ 100 x Km. Equal to kcat[E]tot. vo = initial velocity, linear portion of progress curve where <5% of [S] is consumedG. Ki = equilibrium constant for EI dissociationXXIV. Monitoring Enzyme ReactionsA. Mix E with S in buffered aqueous solutionB. Measure [S] or [P] at set time intervalsC. Plot [S] vs time or [P] vs timeD. Slope of initial straight line = vo or vi Initial velocityE. Always have a unit of time in the denominatorXXV. Repeat over range of substrate concentrationsA. vo=Rate at the start of an enzyme catalyzed reactionXXVI. A kinetic scheme to explain the empirical hyperbolic plot was first proposed by Michaelis and Menten. XXVII. Michaelis-Menten : A Steady State ApproachA. vo = kcat[ES]XXVIII. Michaelis-Menten: A Steady State ApproachA. Key assumptions:1. [P] = 0 at time zero, so that the rate of the reaction depends exclusively on the breakdown of ES and is not influenced by the reverse reaction2. The rate of ES formation equals the rate of ES breakdown; in other words, the reaction is at a steady state. 3. A third assumption is [S] >> [E]tot, so that total [S], which equals free substrate and enzyme-bound substrate, is essentially equal to [S].XXIX. Michaelis-Menten: A Steady State ApproachA. The general expression for the velocity (rate) of the reaction is�_�=�_��� [��]B. and the total enzyme concentration is distributed between E and ES〖[�]〗_���=[�]+[��]C. Dividing the velocity equation by [E]tot where [E] + [ES] is used on the right hand of side, we obtain:�_�/〖[�]〗_��� =(�_��� [��]" " )/([�]+[��])XXX. Michaelis-Menten: A Steady State ApproachA. At the steady state, the rate of [ES] formation, and [ES] consumption are equalB. Rate of formation = k1[E][S] C. Rate of consumption = k-1[ES] + kcat[ES]D. Setting these two rates equal to each then separating rate constants and concentrations[ES]=(�_1 [�][�]" " )/((�_(−1)+�_���)) = ([�][�]" " )/��XXXI. Michaelis-Menten: A Steady State ApproachA. Because the steady state assumption: B. …Now substituting for [ES] inC. From there, cross multiplying kcat and cancelling [E] after factoring this term gives…D. final rearranging, and substituting Etot*kcatwith vmax gives the familiar MM equationXXXII. Michaelis-Menten PlotA. Relates reaction velocity and substrate concentrationB. Vmax[S] vo = ----------- KM + [S]XXXIII. Michaelis-Menten PlotA. Graphical relationship between reaction velocity and substrateconcentrationXXXIV. KcatA. In an enzyme catalyzed reaction, the overall rate of product formation is vo = kcat [ES].B. If all of the enzyme ([E]Tot) molecules are complexed with substrate (excess [S]) then the maximum velocity occurs and vmax = kcat [E]TotC. This can also be written as kcat = Vmax / [E]Tot.D. kcat is called the turnover numberXXXV. KMA. When KM = [S], vo = 1/2 Vmax .B. KM @ k-1 / k1 = Ks (the enzyme-substrate dissociation constant) when kcat value is << either k-1 ).C. The lower the numerical value of KM, the tighter the substrate binding.D. KM is used as a measure of the affinity of E for S.XXXVI. Why determine Km?A. Km and Vmax determination allows us to characterize substrate binding and to compare multiple substrates of the same enzyme.B. Km and Vmax determination allows us to characterize the