Paul D. Adams University of Arkansas Mary K. Campbell Shawn O. Farrell Chapter Six The Behavior of Proteins: Enzymes

Enzyme Catalysis Enzyme: a _____________________ with the exception of some __________ that catalyze their own splicing (Section 10.4), all enzymes are proteins (???) enzymes can increase the rate of a rxn by a factor of up to over an uncatalyzed rxn some enzymes are so specific that they catalyze the rsn of only one stereoisomer; others catalyze a family of similar rxns The rate of a reaction depends on its activation energy,  G° ‡ an enzyme provides an alternative pathway with a ______________________________

Enzyme Catalysis (Cont’d) For a reaction taking place at constant temperature and pressure, e.g., in the body the change in __________________________ is Difference in energies between initial state and final state The change in free energy is related to the equilibrium constant, K eq, for the reaction by

Enzyme Catalysis (Cont’d) Consider the reaction H 2 O 2 → H 2 O + O 2

Temperature dependence of catalysis Temperature can also “catalyze reaction” (increase rate) This is dangerous, why? Increasing temperature will lead to _______________ ______________________

Enzyme Kinetics For the reaction The rate of reaction is given by rate equation ___________________________________________ Where k is a proportionality constant called the ___________________________________________ ______________________________: the sum of the exponents in the rate equation: f+g ______________________________: the sum of the exponents in the rate equation: f+g

Enzyme Kinetics (Cont’d) Consider the reaction Whose rate equation is given by the expression Determined experimentally, not from _______________ The reaction is said to be first order in A, first order in B, and second order overall Consider this reaction of glycogen with phosphate

How Enzymes bind to Substrate In an enzyme-catalyzed reaction ____________________________, S: ____________________________, S: a reactant ______________________: ______________________: the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions

Binding Models Two models have been developed to describe formation of the_____________________ complex __________________ model: __________________ model: substrate binds to that portion of the enzyme with a complementary shape _________________ model: _________________ model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

Two Modes of E-S Complex Formation

Formation of Product

An Example of Enzyme Catalysis ____________________ catalyzes The selective hydrolysis of ___________________ where the ________ is contributed by _____ and ____ It also catalyzes hydrolysis of the ____________ bonds

An Example of Enzyme Catalysis (Cont’d)

Non-Allosteric Enzyme Behavior Point at which the rate of reaction does not change, enzyme is __________________, maximum rate of reaction is reached

ATCase: An Example of Allosteric Behavior ____________ shape - characteristic of __________ Again max velocity reached, but different mechanism

Michaelis-Menten Kinetics Initial rate of an enzyme-catalyzed rxn vs [S]

Michaelis-Menten Model For an enzyme-catalyzed reaction The rates of formation and breakdown of ES are given by these equations At steady state

Michaelis-Menten Model (Cont’d) When ______________is reached, the concentration of free enzyme is the total minus that bound in ES Substituting for the concentration of free enzyme and collecting all rate constants in one term gives K M is called the ____________________________

Michaelis-Menten Model (Cont’d) It is now possible to solve for the concentration of the enzyme-substrate complex, [ES] Or alternately

Michaelis-Menten Model (Cont’d) In the initial stages, formation of product depends only on the _______________________________________________ If substrate concentration is ________________________ is _______________________ [ES] = [E] T Substituting k 2 [E] T = V max into the top equation gives

Michaelis-Menten Model (Cont’d) When _______________ the equation reduces to

Linearizing The Michaelis-Menten Equation V max is difficult to ___________________________________ The equation for a hyperbola Can be transformed into the equation for a ________ by taking __________________________________

Lineweaver-Burk Plot The _______________________ plot has the form y = mx + b, and is the formula for a straight line a plot of 1/V versus 1/[S] will give a straight line with slope of _______________ and y intercept of _______________ __________________________________________ known as a __________________________________________

Lineweaver-Burk Plot (Cont’d) K M is the ________________________________________ the greater the value of K M, the ________ tightly S is bound to E V max is the ___________________________________

Turnover Numbers V max is related to the ___________________________ of enzyme: also called k cat Number of moles of substrate that react to form product _____________________________________________

Enzyme Inhibition ____________ inhibitor: ____________ inhibitor: a substance that binds to an enzyme to inhibit it, but can be released ____________________________ inhibitor: ____________________________ inhibitor: binds to the active (catalytic) site and blocks access to it by substrate _____________________ inhibitor: _____________________ inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation ________________________inhibitor: ________________________inhibitor: a substance that causes inhibition that cannot be reversed usually involves formation or breaking of covalent bonds to or on the enzyme

Competitive Inhibition Substrate competes with inhibitor for the active site; more substrate is required to reach a given reaction velocity We can write a dissociation constant, K I for EI

Competitive Inhibition

In a Lineweaver-Burk plot of 1/V vs 1/[S], the __________________ (and the x intercept) changes but the ______________________ does not change

A Lineweaver-Burke Plot, Competitive Inhibition

Noncompetitive Inhibition (Cont’d) Several equilibria are involved The maximum velocity V max has the form

Noncompetitive Inhibition (Cont’d)

Lineweaver-Burke Plot, Noncompetitive Inhibition Because the inhibitor does not interfere with ______________ to the active site, K M is ______________________ Increasing substrate concentration ____________________ noncompetitive inhibition

Lineweaver-Burke Plot, Noncompetitive Inhibition

Other Types of Inhibition _____________________ - inhibitor can bind to the ES complex but not to free enzyme; V max decreases and K M decreases. __________________ - Similar to noncompetitive, but binding of I affects binding of S and vice versa.