39SectionEnzyme Kinetics81. Produce a reading log for the sections in your text that discuss the Michaelis-Menten equation, including kcat.2. Focus on the derivation of the Michaelis-Menten equation. List and explain the assumptions underlying the Michalis-Menten equation. Provide defi nitions for each term.3. What is equal at equilibrium?4. What is the general expression Keq (the equilibrium constant) in terms of product and reactant concentra-tion?WhyEnzymes catalyze most reactions that occur in biological systems and thus control the rates of these reactions. Examining the kinetics of an enzyme catalyzed reaction helps us learn about how enzymes function. In this activity, you will learn about the kinetic parameters that biochemists use to characterize enzymes and the graphical methods that allow biochemists to obtain kinetic parameters from experimental data.Outcomes1. Be able to articulate and apply what the enzyme parameters of KM, Vmax, kcat and kcat /KM tell us about the enzyme.2. Know the Michaelis-Menten rate equation and be able to utilize the rearranged versions of the Michaelis-Menten equation to determine the values for KM and Vmax.3. Use KM and Vmax values to discuss qualities of enzymes related to their catalytic properties.Plan1. Form enzyme teams. Assign roles of manager, refl ector, recorder, spokesperson.2. Answer the Critical Thinking Questions.3. Share two insights your team made about enzyme kinetics today.Critical Thinking Questions1. Recall your study of equilibria and kinetics from general chemistry. You used equations with upper case K and lower case k during the study of equilibria and kinetics respectively. What do the upper and lower case letters refer to?Foundations of Biochemistry4000.511.522.533.544.5051015[S]. mMVelocity, mM product/minGraph 1Equation 1Equation 2 1Vo=KmVmax[S]+1VmaxE + S 1kESk2⎯ → E + Pk−12. Compare your answers from your assignment and take only two minutes. Discuss the assumptions made during the derivation of the Michaelis-Menten equation. Be sure to consider assumptions pertaining to [ES], rate of product formation, [S], and number of substrates per reaction. 3. Defi ne the term Vmax in your own words4. Solve the Michaelis-Menten equation for KM when V0 = Vmax /2. What does this tell you about the relationship between [substrate] and enzymes with high KM values? With low KM values? Make a generalization.5. a. What is the KM value as you can estimate it from Graph 1? b. On Graph 1, draw a curve that has the same Vmax but a larger KM value.6. Examine Equation 2. When k2 is very small compared to k-1 the KM becomes an approximation of the affi nity of enzymes for substrate. Explain why this approximation makes sense. Does a large KM indicate high or low affi nity of enzyme for substrate?41Section 8 — Enzyme Kinetics7. When k2 is very small compared to k1 and k–1, what process is rate determining in product formation? 8. Many enzyme-catalyzed processes are multi-step reactions. a. In a multi-step reaction, what step determines the overall reaction rate? b. What step in M-M kinetics determines overall reaction rate? c. The term used for the overall rate constant for multi-stp reactions is kcat. Discuss why it makes sense that kcat = k2 in simple Michaelis-Menten reactions.9. In simple M-M kinetics, the units of kcat are s–1. Discuss how the units are consistent with the name “turnover number.” If kcat is large, what does that imply about the enzyme?10. The term effi ciency is often used in describing enzymes. What does it mean for an enzyme to be effi cient?continued on next pageFoundations of Biochemistry4211. How is your answer to question 9 consistent with the term for catalytic effi ciency (kcat /Km)? Discuss the contribution of the terms kcat and KM to the overall term of catalytic effi ciency.12. Discuss the meaning of enzyme reaction mechanism in the context of catalysis. How does the study of enzyme kinetics relate to reaction mechanism?Consider the enzymes in the table below and answer the following questions. Answers should be brief (1-3 sentences).Enzyme KM(M) kcat(s-1)A 9.5 × 10-51.4 × 104B 2.5 × 10-21.0 × 107C 5.0 × 10-68.0 × 1021. Which enzyme has the highest affi nity for substrate? How do you know? 2. Which enzyme converts the most substrate to product in a given period of time? How do you know?43Section 8 — Enzyme Kinetics3. Which enzyme has the highest catalytic effi ciency? How do you know?N|HO|HN−HN|HRCαCβCONCN−HR’Gly 193Ser 195Asp 102His 57C–OOcatalytictriadA diagram of the enzyme active site for chymotrypsin is shown. The amino acids Gly 193, Ser 195, His 57 and Asp 102 form part of the active site. The amino acids Ser 195, His 57 and Asp 102 comprise what is known as the catalytic triad. A portion of a natural peptide substrate from R to R’ is depicted with the residue phenylalanine occupying the hydrophobic pocket of the enzyme.4. Chymotrypsin is a serine protease enzyme. The KM for the reaction of chymotrypsin with N-acetylvaline ethyl ester is 8.8 × 10-2 M, and the KM for the reaction of chymotrypsin with N-acetyltyrosine ethyl ester is 6.6 × 10-4 M. a. Which substrate has a higher apparent affi nity for the enzyme? b. Propose a reason for the difference in affi nity based on the shape of each of the substrates (see active site fi gure, chymotrypsin cleaves on the C-side of aromatic residues). c. Which of the substrates is likely to have a higher Vmax?Foundations of Biochemistry44(This page intentionally left