DOC PREVIEW
UA BIOC 460 - Enzymes - Kinetics

This preview shows page 1-2-3-4 out of 12 pages.

Save
View full document
View full document
Premium Document
Do you want full access? Go Premium and unlock all 12 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 12 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 12 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 12 pages.
Access to all documents
Download any document
Ad free experience
Premium Document
Do you want full access? Go Premium and unlock all 12 pages.
Access to all documents
Download any document
Ad free experience

Unformatted text preview:

BIOC 460, Spring 2008LEC 11, Enzymes - Kinetics 1Lecture 11Enzymes: KineticsReading: Berg, Tymoczko & Stryer, 6th ed., Chapter 8,pp. 216-225Key Concepts• Kinetics is the study of reaction rates (velocities).• Study of enzyme kinetics is useful for measuring– concentration of an enzyme in a mixture (by its catalytic activity),– its purity (specific activity),– its catalytic efficiency and/or specificity for different substrates– comparison of different forms of the same enzyme in different tissues ororganisms,– effects of inhibitors (which can give information about catalyticmechanism, structure of active site, potential therapeutic agents...)• Dependence of velocity on [substrate] is described for many enzymes by theMichaelis-Menten equation:• kinetic parameters:– Km (the Michaelis constant)– kcat (the turnover number, which relates Vmax, the maximum velocity,to [Et], the total active site concentration)– kcat/Km (the catalytic efficiency of the enzyme)– can't be greater than limit imposed by diffusion control, ~108-109 M–1sec–1• Kinetic parameters can be determined graphically by measuring velocity ofenzyme-catalyzed reaction at different concentrations of substrate(Vo vs. [substrate]).BIOC 460, Spring 2008LEC 11, Enzymes - Kinetics 2Learning Objectives• Terminology: active site, enzyme-substrate complex, induced fit, initialvelocity, steady state, Vmax , Km , kcat , turnover number, KES , enzymeefficiency.• Write out a simple Michaelis-Menten kinetic mechanism for an enzyme-catalyzed reaction.• Recognize the Michaelis-Menten equation, and sketch a graph of Vo vs. [S]for an enzyme-catalyzed reaction that illustrates Vmax and Km.• Define Km in terms of the rate constants in the Michaelis-Menten kineticmechanism; give the operational definition of Km that holds no matterwhat the actual kinetic mechanism is for a particular enzyme.• Explain the relationship of kcat to Vmax, and the relationship of Km to KES.• State the units of Km, kcat, and Vmax.• Express the ratio of occupied active sites to total enzyme active sites([ES]/[ET]) in terms of Vo and Vmax. What is the maximum possible value ofthat ratio?• Given a plot of Vo/Vmax vs. [S], find the value of Km from the plot.• What two things is the parameter kcat/ Km used to indicate?– What sets the upper limit for the value of kcat/Km for an enzyme?– What is the approximate range of numerical values for that upper limit ofkcat/Km, with units?REVIEW: How do enzymes reduce activation energy (ΔG‡)?1. by lowering the free energy of the transition state (‡), e.g., bybinding the transition state tightly2. by changing the reaction pathway by which reactants react toform products; e.g., taking a 1-step uncatalyzed reaction andaccomplishing the same result by a different route, with severalintermediate reactions en route.• Each reaction step has its own transition state with its own activation energy (ΔG‡).• If all of the individual steps' ΔG‡s are lower than the activation energy of the uncatalyzed reaction, the overall rate of product formation will be greater in the presence of the catalyst.• The overall rate of the catalyzed reaction is dictated by the slowest step in a multistep reaction. Given a free energy diagram like the one in Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed. (2004) Fig. 6-3 (previous lecture notes), how do you identify the rate-limiting (slowest) step on the reaction coordinate?BIOC 460, Spring 2008LEC 11, Enzymes - Kinetics 3BINDING = the essence of enzyme action!• binding of SUBSTRATE to form an ES COMPLEX• binding of TRANSITION STATE more tightly than the substrate• Binding occurs at ACTIVE SITE of enzyme.• Subsequent chemical events can then occur.• Active site:–relatively small part of whole enzyme structure–3-dimensional cleft with participating components from different parts of primary structure–water often excluded so substrates and intermediates are in non-aqueous environment (unless H2O is a reactant)• Binding uses multiple weak interactions:1. hydrogen bonds2. salt links3. van der Waals interactions4. hydrophobic effectLysozyme (residues from different parts of AA sequencecome together in active site)Berg et al., Fig. 8-7BIOC 460, Spring 2008LEC 11, Enzymes - Kinetics 4Specificity of binding• depends on active site crevice being sterically and chemicallycomplementary to groups it is binding (best complementarity may bepresent in ES complex but NOT in free enzyme -- induced fit)• Enzymes flexible -- conformational changes can occur when substratebinds during the reaction, to get maximal complementarity to thetransition state.• induced fit: conformational changes giving tighter binding in a newconformation• For many (probably most) enzymes, the active site assumes shape complementary to S only when S is bound.Berg et al., Fig. 8-10Why study enzyme kinetics (reaction rates)?• measurement of velocity = reaction rate• compare enzymes under different conditions, or from differenttissues or organisms– understand how differences relate to physiology/function of organism– e.g., physiological reason for different Km values for hexokinase vs.glucokinase (discussed later in course)• compare activity of same enzyme with different substrates (understandspecificity)• measure amount or concentration of one enzyme in a mixture by itsactivity• measure enzyme purity (specific activity = amount of activity/amount ofprotein)• study/distinguish different types of inhibitors– info about enzyme active sites and reaction mechanism– development of specific drugs (enzyme inhibitors)BIOC 460, Spring 2008LEC 11, Enzymes - Kinetics 5Simple Enzyme-Catalyzed Reaction:• Measurement of velocity:– V = rate of appearance of product = change in [P] per unit time– units of velocity (V)? _______________Plot of [P] vs. time• Experimentally, forward velocity VF = slope of plot of [P] vs. time because VF = kF[S] = Δ[P]/Δ tBerg et al., Fig. 8.11(slightly modified)Determining initial velocity, Vo• Why we measure initial velocity, Vo, the slope of [P] vs. [time] atvery early time after mixing enzyme with substrate• Problem:– As S is converted to P, concentration of S decreases, so forwardvelocity gets slower and slower.– Furthermore, as [P] increases, rate of reverse reaction (P --> S)becomes significant, and eventually, when VF =


View Full Document

UA BIOC 460 - Enzymes - Kinetics

Documents in this Course
Exam 4

Exam 4

3 pages

Enzymes

Enzymes

4 pages

Enzymes

Enzymes

14 pages

Exam 4

Exam 4

6 pages

Enzymes

Enzymes

10 pages

Enzymes

Enzymes

5 pages

Enzymes

Enzymes

19 pages

Load more
Download Enzymes - Kinetics
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Enzymes - Kinetics and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Enzymes - Kinetics 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?