What is an enzyme? Lecture 12: Enzymes & Kinetics I Introduction to Enzymes and Kinetics. Margaret A. Daugherty Fall 2004 KEY FEATURES OF ENZYMES

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1 Lecture 12: Enzymes & Kinetics I Introduction to Enzymes and Kinetics Margaret A. Daugherty Fall 2004 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 CATALYTIC POWER: ratio of catalyzed reaction rate to uncatalyzed reaction rate; enzymes accelerate reactions as much as ; important to note that they do this under physiological conditions (ph 7, 37C, H 2 0) KEY FEATURES OF ENZYMES 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.

2 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: breaks down liver glycogen stores to glucose 1). Non-covalent interactions modulate response to fuel needs: High fuel state: Enzyme off high ATP high Glucose high G6P Low fuel state: Enzyme on high AMP 2). Covalent modification Stress situation! Molecule always 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 ); Transition State and Free Energy Consider a reversible reaction A<----->B Transition State and Free Energy Transition state theory provides information on G and says that G 1 is smaller than G -1, thus reaction favors formation of B Thermodynamics tells it will proceed in the direction 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

3 Catalytic Reactions Six Major Classes of Enzymes A B 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 Do not raise energy of A Catalysts (e.g. enzymes) act by lowering the transition state free energy for the reaction being catalyzed. Oxidoreductases Oxidation-reduction reactions (addition or removal of hydrogen atoms from many chemical substituents) Example: dehydrogenases Transferases Transfer of functional groups between donor and acceptor molecules; Example: kinases Oxidases, oxygenases, reductases, peroxidses & hydroxylases Groups: amino, carboxyl, carbonyl, methyl, phosphoryl and acyl (RC=0)

4 Hydrolases Cleavage by hydrolysis reactions (adding H 2 O across a bond); Example: the proteases Lyases Group elimination or addition to double bonds; Frequently H 2 O, NH 3 or CO 2 ; Example: pyruvate decarboxylate (esterases, phosphatases & peptidases) (hydratases, dehydratases, deaminases, synthases) Isomerases Ligases Isomerization reactions (intramolecular rearrangements); Bond formation by condensation of two groups coupled to ATP hydrolysis; Example: alanine racemase Example: polymerases Epimerases: catalyze interconversion of asymmetric carbon atoms Mutases: catalyze intramolecular transfer of functional groups (synthetases, carboxylases)

5 COENZYMES: ENZYMES NEED HELP CHEMICAL KINETICS FIRST ORDER REACTIONS & THE RATE CONSTANT A k 1 B k 1 = rate constant for the reaction (units = sec -1 ) The rate law: V = d[b]/dt or -d[a]/dt or Recall our definitions of apoprotein, prosthetic group, holoenzyme) V = -d[a]/dt = k[a] CHEMICAL KINETICS SECOND ORDER REACTIONS & THE RATE CONSTANT SIMPLE EXAMPLE k A + B k k = (moles/l) -1 sec -1 2A A 2 Rate Law: C + D V = -d[a] 2 /dt = k[a] 2 or V = -d[a]/dt = -d[b]dt = d[c]/dt = d[d]/dt = k[a][b] 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

6 Review 1). Enzymes are protein catalysts that speed up biological reactions by as much as ). 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.