Lecture 6/7 - Chapter 6: The Behavior of Proteins: Enzymes

Enzyme Catalysis

  • Enzyme: biological catalyst

    • all enzymes are proteins (exception: some RNAs)

    • can increase the rate of the reaction

    • can be specific to only one stereoisomer

    • rate of reaction depends on activation energy (∆Gº)

      • enzyme provides alternative pathway to decrease ∆Gº

Temperature Dependence of Catalysis

  • temperature can increase rate (catalyze reaction)

    • dangerous as increasing temperature will lead to protein denaturation

Enzymes

Specificity

  • convert a select group of substrates (reactants) to a select group of products

  • enzymatic reactions proceed without undesired side reactions

    • phenylalanine hydroxylase - selective of stereochemistry

Active Site

  • specific site on/in the enzyme where substrate binds and catalysis takes place

    • E + S ← → ES (enzyme-substrate complex)

    • enzymes act by binding substrates

Binding Models for Formation of ES Complex

  • Lock and key: substrate binds to portion of enzyme with complementary shape

    • same conformation

  • Induced fit: binding of substrate induces change in conformation of the enzyme that results in a complementary fit

    • enzyme undergoes conformational change

Catalytic Power

  • ratio of enzyme-catalyzed rate to the uncatalyzed rate (rate enhancement)

Reaction Kinetics

  • A + B → P

  • Rate = -∆[A]/∆t = -∆[B]/∆t = ∆[P]/∆t

  • Rate = k[A]^f [B]g

    • k = rate constant

    • order = sum of exponents in rate equation

  • rate can be independent of concentration of reactants

    • depends on other factors (catalyst)

    • enzyme catalyzed reactions can exhibit zero order kinetics when the enzyme’s active site is saturated with substrate

Chymotrypsin Catalysis

  • selective hydrolysis of peptide bonds where carbon is contributed by Phe and Tyr

    • catalyzes hydrolysis of ester bonds

  • initial velocity vs. initial substrate

    • point which the rate of reaction does not change, enzyme is saturated, maximum rate of reaction is reached (zero order)

Regulation

Allosteric Control

  • effect produced on the activity of one part of an enzyme by the binding of an effector to a different part of an enzyme

  • v = Vmax[S]^n/K’ +[S]^n

    • n= hill number (minimum number of active sites)

    • K’= average Km, compromises Km values for the binding of each substrate to an active site

    • K0.5 = [S]0.5 = substrate concentration yielding ½ Vmax

      • K0.5 = n log [S]0.5

Michaelis-Menten Kinetics

  • initial rate of an enzyme-catalyzed reaction vs. substrate concentration

    • E + S ←(k-1) (k1)→ ES (k2)→ P

    • rate of formation of ES = k1[E][S]

    • rate of breakdown of ES = k-1[ES] + k2[ES]

  • at the steady state:

    • k1[E][S] = k-1[ES] + k2[ES]

    • [E] = [E]Total - [ES]

      • ([E]Total - [ES])[S]/[ES] = k-1 +k2/k1 = Km

      • solve for the concentration of the enzyme-substrate complex

        • [ES] = [E]Total[S]/ Km + [S]

  • v0 is determined by the breakdown of ES to form product

    • v0 = k2[ES]

    • v0 = vmax = k2[E]Total

      • v0 = vmax [S]/Km + [S]

      • when [S] = Km the equation reduces to vmax/2

  • vmax = k2[E]Total = kcat[E]Total

    • k2 = kcat

    • kcat is the turnover number (number of substrate molecules converted into product by an enzyme per unit time, when the enzyme is fully saturated with substrate)

  • kcat/Km is second order rate for conversion of S to P at low [S]

    • v = kcat/Km [E]Total[S]

      • “specificity constant”

Determination of Kinetic Parameters

  • nonlinear Michaelis-Menten plot should be used to calculate parameters Km and Vmax

  • linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition

    • lineweaver-burke plot: 1/v vs. 1/[S]

      • 1/v = (Km/vmax)(1/[S]) + (1/vmax)

      • y = m x + b

    • eadie-hofstee plot: v vs. v/[S]

    • hanes-wolfe plot: [S]/v vs. [S]

Enzyme Inhibition

  • inhibitors: compounds that decrease the rate of an enzyme catalyzed reaction

Reversible Inhibitor

  • substance that binds to an enzyme to inhibit it, but can be released

    • often structural analogs of substrates or products

    • often used as drugs to slow down a specific enzyme

    • can bind to the free enzyme and prevent binding of substrate

    • can bind to ES and prevent the reaction

Competitive Inhibitor

  • binds to the active site and blocks access to it

  • substrate must compete with inhibitor for the active site

    • more substrate is required to reach a given reaction velocity

    • E + I ←(k-3) (k3)→ EI

      • 1/v = Km/Vmax(1 + [I]/KI)1/S + 1/Vmax

      • slope and x intercept changes but the y intercept does not

Noncompetitive Inhibitor

  • binds to site other than the active site

  • inhibits the enzyme by changing its conformation

Irreversible Inhibitor

  • substance that causes inhibition that cannot be reversed

  • usually involves formation or breaking of covalent bonds

    • can permanently shut off enzyme

    • often powerful toxins but may be used as drugs

  • inhibitor irreversibly binds to an enzyme or causes irreversible damage to the enzyme upon binding

    • time-dependent loss of enzyme activity

  • *penicillin*

Other Types of Inhibition

Non-Competitive (mixed)

  • inhibitor can bind to both E and the ES complex.

  • Vmax decreases

  • Km can increase or decrease

Uncompetitive

  • inhibitor can bind to the ES complex

  • Vmax decreases

  • Km decreases