Lehninger Principles of Biochemistry David L. Nelson and Michael M. Cox Lehninger Principles of Biochemistry Fourth Edition Chapter 6: Enzymes Copyright © 2004 by W. H. Freeman & Company

Definitions Substrate: Species that is acted upon by enzyme Prosthetic group: Coenzyme or cofactor tightly (permanently) bound to enzyme Holoenzyme: Enzyme + prosthetic group Apoenzyme (apoprotein): Enzyme – prosthetic group Active Site: Pocket within enzyme in which reaction occurs Ground State: Starting point for forward/backward reactions; lowest ENERGY Transition State: Top of energy “hill”; forward and reverse reactions are equally likely

Thermodynamics of Enzyme Catalyzed Reactions G: Free energy change G°: Standard free energy change (T = 298K; P = 1atm; Concentration = 1M) G°: Biochemical standard free energy change (As above, but pH 7) Relationship between free energy change and equilibrium constant K: G° = -RT ln K G‡ = activation energy

Kinetics Overall rate of reaction is determined by the RATE LIMITING STEP (slowest step) G° involves equilibrium, but G‡ involves reaction rate Rate equation (rate law): 1st order rate: (V) = k[S], where V and S are velocity and substrate concentration, respectively 2nd order rate: V = k[S1][S2] Rate constant (k) is related to G‡ by What is the mathematical relationship between G‡ and k?

How do they work? Covalent interactions, though transient and short-lived, activate chemical groups for reaction Non-covalent interactions: Formation of weak bonds between E and S release energy (GB) Free energy from the quantity of these weak interactions is used to catalyze reactions and Weak interactions are optimized by enzyme-transition state binding (not enzyme-substate)

What contributes to G‡? How are these issues overcome? Reduction in Entropy Binding energy used to hold substrate in place for reaction Reduction in hydration shell Binding between E and S compensate for bonds lost between E and water Conformational change in substrate Binding to TS optimizes interactions Conformational change in enzyme Change in protein shape IMPROVES/INCREASES interactions: INDUCED FIT

Reduction of entropy by enzyme

Types of Catalysis Acid-base: Charged intermediates are stabilized

Types of Catalysis Acid-base: Charged intermediates are stabilized Specific acid-base: Uses H+ or OH- from solution General acid-base: H+ transfer from amino acid proton donor Q: Which amino acids are most likely to act as general acids or bases? Covalent: Formation of transient, covalently attached intermediate provides alternate route to products, lower in activation energy Can be E-S or C-S (C = cofactor)

Types of Catalysis Acid-base: Charged intermediates are stabilized Specific acid-base: Uses H+ or OH- from solution General acid-base: H+ transfer from amino acid proton donor Q: Which amino acids are most likely to act as general acids or bases? Covalent: Formation of transient, covalently attached intermediate provides alternate route to products, lower in activation energy Can be E-S or C-S (C = cofactor) Metal ion Ionic interactions stabilize E-S Electron donation for REDOX

Enzyme Kinetics: Michaelis-Menten Kinetics

Enzyme Kinetics Rate of reaction is sensitive to [E]

G. P. Royer1 and Sheldon E. Broedel, Jr G. P. Royer1 and Sheldon E. Broedel, Jr.2 Buford Biomedical, Frederick, MD1 Athena Environmental Sciences, Inc., Baltimore, MD2

Rate of reaction is sensitive to [E] As reaction proceeds, [S] decreases while [P] increases In early part of reaction, rates are linear. We call this rate “initial velocity (v0)

If we plot v0 vs. [S], the rate plateaus at a maximum If we plot v0 vs. [S], the rate plateaus at a maximum. This “maximum rate” is called vmax.

How can we study enzyme kinetics? E = enzyme S = substrate ES = enzyme-substrate complex (Michaelis complex) 2 Simplifying Assumptions: Step 1 is in equilibrium because k-1 >> k2 d[ES]/dt = 0

Bisubstrate Reactions Some enzymatic reactions require more than one substrate and produce more than one product Substrates are named A, B, C, D…in order in which they add to enzyme Products are named P, Q, R, S… in order in which they leave the enzyme The number of substrates and products involved in a reaction is indicated as Uni, Bi, Ter, Quad Example: A reaction with 2 substrates and 2 products is called “Bi Bi”

Types of Bisubstrate Mechanisms I. Sequential Reactions Ordered Reactions: Order of substrate addition is compulsory A B P Q k1 k-1 k2 k-2 k4 k-4 k5 k-5 k3 E EA EAB k-3 EPQ EQ E

Types of Bisubstrate Mechanisms I. Sequential Reactions Random Reactions: Order of substrate addition is NOT compulsory

Types of Bisubstrate Mechanisms II. Ping Pong Reactions Some products are released before all substrates are added E Q P B A EA-FP F FB-EQ

Enzyme Inhibition

Competitive Inhibition Inhibitor (I) competes with S for binding to active site

Uncompetitive Inhibition

Mixed Inhibition

pH Dependence of Enzyme Activity

Chymotrypsin: An example of catalysis

Other Examples of Enzyme Catalytic Mechanisms See Hexokinase, Lysozyme, Enolase

Enzyme Regulation Allosterism: Binding of inhibitors or activators cause conformational changes, altering enzyme activity and kinetics

Feedback inhibition

Covalent Modification

Cleavage of Inactive Enzyme Precursor: Zymogens