Lecture 12: Enzymes & Kinetics I Introduction to Enzymes and Kinetics Margaret A. Daugherty Fall 2003 ENZYMES: Why, what, when, where, how? All but the who! What: proteins that exert kinetic control over biological reactions Why: We need to have chemical reactions occur on a biologiclly relevant time scale (recall the example from lecture 1) When: At appropriate times during metabolic processes (brings us to the idea of biological regulation - lecture 16). Where: Where ever chemistry needs to be accomplished in the body How: Through the bringing together of reactive groups on the enzyme with the substrates that need to have chemistry performed on them. THERMODYNAMIC POTENTIALITY: reaction is strongly exergonic; but doesn t occur under normal conditions. It needs help. Enzymes provide the help - they lower the activation energy necessary to have a reaction go to completion What is an enzyme? General Properties Mostly proteins, but some are actually RNAs Biological catalysts Higher reaction rates Milder reaction conditions Great reaction specificity Capacity for regulation Not changed or used up after a reaction Nomenclature: frequently add -ase
KEY FEATURES OF ENZYMES KEY FEATURES OF ENZYMES CATALYTIC POWER: ratio of catalyzed reaction rate to uncatalyzed reaction rate; enzymes accelerate reactions as much as 10 20 ; important to note that they do this under physiological conditions (ph 7, 37C, H 2 0) SPECIFICITY: Enzymes are selective about their substrates (also called ligands, reactants) and the chemistry they carry out (active sites are specialized for both the reactant and the chemistry). There are no wasteful by-products. KEY FEATURES OF ENZYMES REGULATION: Enzymes should only function when needed. They are exquisitely regulated at the level of DNA, by interactions with inhibitors and activators, by product feed-back inhibition. Glycogen Phosphorylase 1). Response to fuel needs: High fuel state: Enzyme off high ATP high Glucose high G6P Low fuel state: Enzyme on high AMP low ATP 2). Covalent modification Stress situation! Molecule on! Enzymes as Catalysts the take home points Enzymes DO NOT change the equilibrium constant of a reaction Enzymes DO NOT alter the amount of energy consumed or liberated in a reaction ( H); Enzymes DO increase the rates of reactions that are otherwise impossible; Enzymes DO decrease the activation energy ( G );
Enzymes Enzymes are protein catalysts Bind the substrates Lower the activation energy Directly promote the catalytic events Transition State and Free Energy Consider a reversible reaction A<----->B Catalyst enhancement Non-enzymatic (metals) Enzymatic Rate 10 2-10 4 fold up to 10 20 fold How much is 10 20 fold? - with a catalyst, the reaction takes place in 1 sec - without a catalyst, 3 x 10 12 years! Thermodynamics tells it will proceed in the direction of B Transition State and Free Energy Catalytic Reactions Transition state theory provides information on G and says that G 1 is smaller than G -1, thus reaction favors formation of B G = Free Energy of Activation determines the rate of reaction k= Ae - G /RT (Arrhenius Equation) G : rate is proportional to # of molecules that have this energy A B Do not raise energy of A Catalysts (e.g. enzymes) act by lowering the transition state free energy for the reaction being catalyzed.
Six Major Classes of Enzymes Oxidoreductases: oxidation-reduction reactions Transferases: transfer of functional groups Hydrolases: cleavage of bonds by hydrolysis Lyases: group elimination to form double bonds Isomerases: isomerization (simplest) Ligases: bond formation between 2 substrates Oxidoreductases Oxidation-reduction reactions (addition or removal of hydrogen atoms from many chemical substituents) Example: dehydrogenases Oxidases, oxygenases, reductases, peroxidses & hydroxylases Transferases Transfer of functional groups between donor and acceptor molecules; Example: kinases Hydrolases Cleavage by hydrolysis reactions (adding H 2 O across a bond); Example: the proteases Groups: amino, carboxyl, carbonyl, methyl, phosphoryl and acyl (RC=0) (esterases, phosphatases & peptidases)
Lyases Group elimination or addition to double bonds; Frequently H 2 O, NH 3 or CO 2 ; Isomerases Isomerization reactions (intramolecular rearrangements); Example: alanine racemase Example: pyruvate decarboxylate (hydratases, dehydratases, deaminases, synthases) Epimerases: catalyze interconversion of asymmetric carbon atoms Mutases: catalyze intramolecular transfer of functional groups Ligases Bond formation by condensation of two groups coupled to ATP hydrolysis; Example: polymerases Some examples: To what class of an enzyme do the following enzymes belong? (synthetases, carboxylases)
COENZYMES: ENZYMES NEED HELP CHEMICAL KINETICS FIRST ORDER REACTIONS & THE RATE CONSTANT A k 1 k -1 B k 1 = rate constant for the forward reaction k -1 = rate constant for the reverse reaction (units = sec -1 ) The rate law: V = d[b]/dt or -d[a]/dt At equilibrium V = k 1 [A] - k -1 [B] = 0 Recall our definitions of apoprotein, prosthetic group, holoenzyme) or V = -d[a]/dt = k[a] k 1 k -1 = [B] eq [A] eq = Keq CHEMICAL KINETICS SECOND ORDER REACTIONS & THE RATE CONSTANT SIMPLE EXAMPLE A + B k 2 k -2 k 2 C + D 2A A 2 k -2 k 2 = (moles/l)-1 sec -1 Enzymes and Equilibrium k f A B k r [B] [A] k f K = = = = 100 k r k f = 10-4 s -1 k r = 10-6 s -1 10-4 10-6 Rate Law: At equilibrium V = k 2 [A] 2 - k -2 [A 2 ] = 0 Therefore, at equilibrium the [B] is 100 times [A] V = -d[a] 2 /dt = k 2 [A] 2 k 2 k -2 = [A 2 ] eq [A] 2 eq = Keq Note: Enzymes accelerate the attainment of equilibria, but they DO NOT shift their positions!
Enzyme Reactions E + S ES ES* EP E + P E = enzyme ES = enzyme-substrate complex ES* = enzyme/transition state complex EP = enzyme product complex P = product Physically interact with their substrates to effect catalysis; Substrates bind to the enzyme s active site Review 1). Enzymes are protein catalysts that speed up biological reactions by as much as 10 20. 2). Enzymes work by reducing the G, not by altering the equilibrium constant. 3). G, is the additional energy that substrates have to have above and beyond their intrinsic energy to reach the transition state. By reducing the G, there are molecules that can reach the transition state. 4). The transition state represent a barrier that reactants must go through. Once they reach the transition state, there is a high probability that the reaction will proceed to completion. In order to do chemistry, the reactants are usually distorted or strained in this state. 5). Three key features of enzymes are their catalytic power, their specificity for substrates and the chemistry they perform and their ability to be regulated. 6). There are 6 major classes of enzymes (be familiar with them). 7). We can use the ideas of chemical kinetics to understand enzyme kinetics. 8). Enzymes physically interact with their substrates.