Enzymes are specialized proteins that catalyze biological reactions, accelerating reaction rates as much as fold over uncatalyzed rates.

Transcription

1 Chapters 10 and 11 Enzymes Enzymes are specialized proteins that catalyze biological reactions, accelerating reaction rates as much as fold over uncatalyzed rates. 6 major classes of enzymes: 1) Oxidoreductases Catalyze oxidation-reduction reactions 2) Transferases: Transfers functional groups between donors and acceptors kinases: transfer phosphate groups 3) Hydrolyases: Special class of transferases in which the donor group is water. Proteolytic enzymes are a special class of hydrolyases called peptidases. 4) Lyases: Add or remove elements of H 2 O, NH 3, CO 2 an example is a decarboxylase: removes CO 2 from amino acids an example is a dehydatase: remove H 2 O in a dehydration reaction 5) Isomerases: Catalyze isomerization of several types: cis-trans, aldose-ketose, etc. A mutase is an example in which there is an intramolecular transfer of a group.

2 6) Ligases: Involved in synthetic reactions where 2 molecules are joined together in an endothermic reaction. Require energy (ATP) Coenzymes: Many enzymes require coenzymes or cofactors in order to carry out their reaction. 1) ATP adenosine triphosphate Made up of an adenine base + ribose sugar + triphosphate group connected to the sugar. Can be a phosphate donor (kinase) or an energy source (breaking off a phosphate group releases energy). 2) NAD and NADP NAD nicotinamide adenine dinucleotide Used as an electron donor or acceptor in an oxidoreductase reaction, NAD + + H 2 NADH + H + Ox. Red.

3 Can be used to shuttle electrons between different substrates and acceptors. Thus, the reduced form acts as an electron pool. The nicotinamide ring is the active center of this molecule. 3) FAD and FMN forms of riboflavin FAD flavin adenine dinucleotide Functions in redox reactions by accepting and donating 1 or 2 electrons in the flavin ring. 4) Metal Cofactors Metals are required as cofactors in about 2/3 of all enzymes. Metals can act as Lewis acids and by various modes of chelate formation. Lewis acid- transition metal like Zn, Fe, Mn, Cu which have empty d orbitals to act as electron sinks. In addition, the binding of a metal to an enzyme may stabilize the active conformation or also induce the formation of a binding site or active site. Enzyme Kinetics: Review: Kinetics is the study of the rate of change of reactants to products. R P Rate= -ΔR= ΔP = ν = k[r] k= rate constant Δt Δt Example: A + B C + D Rate equations are experimentally determined. If rate = k[a], then the reaction is first order with respect to A, and doubling the concentration of A will double the reaction rate. The concentration of B has no effect on the rate. If rate = k[a][b], then the reaction is first order with respect to A, first order with respect to B, and second order overall. If rate = k[a][b], and we use a huge excess of B in the reaction, then the reaction rate only depends on [A] and this is a pseudo first order reaction.

4 In any reaction, certain conditions must be achieved for the reaction to occur. The reacting molecules must (1) collide together in the (2) correct orientation for reaction and (3) with enough energy for the reaction to occur. The transition state sits at the apex of the energy diagram above, and the energy required to reach this state is the activation energy Ea. We can accelerate the rate of this reaction by either raising the temperature of the reaction (not a real option in the body) or by the addition of a catalyst. A catalyst is regenerated after each reaction cycle and has no effect on the overall free energy of the reaction. Enzymes: k 1 k 3 E + S ES E + P k 2 k 3 =k cat catalytic constant turnover number, the number of catalytic events/second/enzyme The initial velocity, ν o, depends on the amount of substrate present and enzyme concentration. A plot of ν o versus substrate concentration would yield the following plot: Figure 10.7 We want to develop a rate equation that relates the velocity of the reaction (rate) to the substrate concentration. Michaelis-Menten Equation: This rate equation is based upon three assumptions: (1) The ES complex is in a steady state, (2) when [S] is high, all of E is converted to ES, and (3) if all of E is in ES, the rate of the formation of products will be maximized. Vmax= k 3 [ES] Km= breakdown (ES) = k2+k3 Formation (ES) k1 Steady state allows us to balance the formation of ES and breakdown of ES v f =k 1 [E][S] v b =k 2 [ES] +k 3 [ES]=[ES](k 2 +k 3 ) See derivation on pages 328 and 329 v o = V max [S] Michaelis-Menten equation K m + [S] K m is the substrate concentration when v o = ½ V max Lineweaver-Burke Plot (double reciprocal plot) Taking the reciprocal of the Michaelis-Menten equation and rearranging gives the equation: 1/v o = (K m /V max )(1/[S]) + 1/V max

5 y = m x + b equation for a straight line A plot of 1/v o versus 1/[S] gives a straight line with a sloe of K m /V max, a y-intercept of 1/V max, and an x- intercept of -1/K m. See figure 10.9 Enzyme Inhibition: Inhibitor A compound that binds to an enzyme and interferes with its activity by preventing either substrate binding of S to E or preventing the breakdown of ES to E and P. I) Reversible Inhibitors A) Competitive Inhibitor- most commonly encountered This inhibitor binds to E and prevents S from binding. It is a competition between I and S to see which will bind to E. E + S ES E + P If I binds to E, then k 1 + Since K m =(k 2 +k 3 )/k 1, Km I Also V max =k 3 [ES] k 3 is unaffected, so V max is unchanged The effects of a competitive inhibitor can be overcome by adding EI more S to the reaction Lineweaver-Burke plot B) Uncompetitive Inhibitor This inhibitor blocks the breakdown of ES to product. E + S ES E + P Here, both k 2 and k 3, but k 1 is not affected + V max =k 3 [ES], so Vmax I K m =(k 2 +k 3 )/k 1, K m ESI Lineweaver-Burke plot C) Mixed Inhibitor Can bind to either E or ES forming an inactive ESI complex That will not lead to the production of P. Does not bind in the substrate binding site. E + S ES E + P Here, k 1, k 2, and k 3 all decrease + + V max =k 3 [ES], so Vmax I I K m =(k 2 +k 3 )/k 1, so K m remains virtually unchanged This inhibitor can be reversed by exhaustive dialysis of the EI ESI inhibited enzyme. Lineweaver-Burke

6 II) Irreversible This inhibitor forms a stable covalent bond with the enzyme molecule irreversible removing active molecules. The Lineweaver-Burke plot looks like a mixed plot. Can use these inhibitors to test which amino acids are important in an enzymatic reaction. An example of an irreversible inhibitor is nerve gas DFP (diisopropyl fluorophosphates). See Serine protease notes. DFP irreversibly inhibits serine proteases, especially serine esterase acetylcholinesterase which catalyzes the hydrolysis of the neurotransmitter acetylcholine to restore the nerve cells to their resting position. When inhibited, the nerve cell can not reset, and you become paralyzed. Another example is penicillin. Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidase, which catalyzes a critical step in bacterial cell wall synthesis. Inhibitors as Drugs: Many drugs have been designed to inhibit a specific enzyme in a metabolic pathway. Most are competitive inhibitors because these are the easiest to design. 1) Sulfa Drugs (competitive inhibitors) Sulfanilamide is an antibacterial agent because of its competition with para-aminobenzoic acid (PABA) which is required for bacterial growth. Sulfanilamide PABA Bacteria must make folic acid in order to replicate its DNA (can not absorb). PABA intermediate folate Sulfanilamide binds to enzyme and blocks the production of folate. Bacteria is starved of folate and can not grow and divide. This drug does not hurt the host because we can absorb folate from food. 2) Methotrexate: One type of chemotherapy drug. An inhibitor of the coenzyme folic acid. Here methotrexate blocks the binding of folate to the enzyme which results in the inability to synthesize RNA and DNA. Since can not make new DNA, the cancer cell can not multiply and divide. The bad news is that all rapidly dividing cells are affected (hair, stomach lining, etc)

7 2) Aspirin and Ibuprofen Both of these inhibit the biosynthesis of prostaglandins by inhibiting the activity of a key enzyme in the biosynthetic pathway. Prostaglandins are known to enhance inflammation in animal tissues. Aspirin is an irreversible inhibitor that inhibits the first step in the biosynthesis, while ibuprofen is a competitive inhibitor. Multisubstrate Reactions: 1) Ping-Pong mechanism Form a modified enzyme intermediate, E*. E + A EA E* + B E*B E + P 2 P 1 Here, B will only bind to E* not E; therefore, A must bind, react, and modify the structure of E in order for B to be able to bind. 2) Sequential mechanism Both A and B must bind before any product is made. E + A EA + B EAB E + P 1 + P 2 An oxidoreductase follows this mechanism, where NADH must bind and then the substrate binds. Allosteric Control Ligand- any molecule that is bound to a macromolecule- can be activators, inhibitors, substrates. Modulators Change enzymatic activity but themselves remain unchanged. Enzymes that respond to modulators have allosteric sites different from substrate binding sites. Positive allosteric effect-bind to an activator site, Negative allosteric effect bind to an inhibitory site. Any time a ligand binds to an enzyme, the 3-D conformation of the enzyme is altered. Classes: K class alters K m : If K m then inhibitor If K m then activator V class alters V max : If V max then inhibitor If V max then activator An enzyme can have separate catalytic and regulatory subunits. Enzyme Regulation 1) Regulate amount of enzyme by a change in the de novo synthesis of the enzyme 2) Modulate enzyme activity with activators, inhibitors, and through covalent modifications.

8 An example of a covalent modification would be phosphorylation. Can phosphorylate an S, T, or Y amino acid with a kinase. Phosphorylation would be adding a phosphate group ligand which can modify the 3- D structure of the enzyme resulting in the activation or inactivation of that enzyme. This process is reversible with a phosphorylase removing the phosphate group. 3) Separate the enzyme from substrates and control access of S to E by compartmentalizing the enzyme. Substrate Binding Site The specificity of the enzyme for substrate resides in the substrate-binding site. The 3-D structure of the enzyme is folded in such a way as to create a region that has the correct molecular dimension (size), appropriate topology (shape), and optimal alignment of amino acids (for non covalent interactions) to accommodate a specific substrate. Enzymes are conformationally dynamic molecules, and the binding of a substrate by an enzyme is an interactive process. Both the shape of the enzyme binding site and the substrate can be modified upon binding. This is known as induced fit. Enzymes can distinguish between isomers (ex:d and L forms of an amino acid) and conformation (ex: chair, boat). Active Site The specificity of enzyme reaction rests in the active site and the amino acids that participate in the bondmaking and bond-breaking reaction. The binding of S to E can result in a change in the orientation of the active site, thus activating the enzyme. Mechanism of Catalysis All chemical reactions have a potential energy barrier and thus an activation energy. Enzymes lower the activation energies of reactions. This can be accomplished in a variety of ways, as exemplified by the following: 1) Acid-Base Catalysis The body does not have free H+ or OH-. Therefore, the acidic amino acids (D, E) are used as acids and the basic amino acids (H, R, K) are used as bases in acid and base catalyzed reactions. These amino acids work best when the ph is at or near the pka of the amino acid side chain. Remember that the environment around the amino acid affects its pka. ph is very important in these reactions. His is most often the most effective acid or base because the pka of its side chain is ) Substrate-strain The binding of the substrate to the binding site causes a strain in the substrate resulting in a less stable, higher energy molecule which is closer in energy to the transition state. 3) Covalent Catalysis Attack of a nucleophilic (-) or an electrophilic (+) group in the enzyme active site upon substrate creates a covalent bond between E and S. 4) Proximity Enzymes bring 2 reactants together with the correct geometric orientation for the reaction to occur. 5) Metal Ion Catalysis Metals can act as electrophilic catalysts, stabilizing the increased electron density or negative charge that can develop during reactions. A metal ion can also provide a powerful nucleophile at neutral ph. Serine Proteases This is a family of enzymes that utilize a single, uniquely activated Ser residue to catalytically hydrolyze peptide bonds. This family includes trypsin, chymotrypsin and elastase (digestive enzymes secreted in the pancreas and secreted into the digestive tract) Thrombin (crucial enzyme in the blood-clotting cascade), plasmin (breaks down the fibrin polymers of blood clots), and others. Trypsin cleaves peptides on the carbonyl side of R and K residues. Chymotrypsin cleaves on the carbonyl side of aromatic residues (F, W, and Y)

9 Elastase is not as specific and cleaves on the carbonyl side of small neutral residues. Can identify which Ser is active by the irreversible reaction of its O- group with DFP (diisopropyl fluorophosphate). Ő - Ser Proteases are synthesized in a precursor, inactive form called a zymogen. The zymogen form is designated with a pro- at the beginning of the enzyme name (prothrombin) or an -ogen at the end (trypsinogen). Active Site: The enzyme folds into a 3-D structure that forms a catalytic triad- His, Asp, and Ser- at the active site. Asp-forms H bonds with the His residue locking it in place, and increasing the basicity of the His. His- Must be in its basic form- pulls the H from the OH group of Ser, thus creating the better nucleophile O - Ser- The activated O- group performs a nucleophilic attack on the amide bond of a peptide in the first step of the mechanism. See page 378 in the text for a detailed mechanism. Substrate Binding Site: There is a substrate binding pocket that resides beside the catalytic triad which give each serine protease its substrate specificity. For example, the pocket in chymotrypsin is large and contains hydrophobic residues to create hydrophobic noncovalent interaction with F and W side chains. The pocket in trypsin contains an Asp residue to stabilize the positive charge of R and K residues. Serine Proteases in Tumor Cell Metastasis: Urokinase is believed to be required for a tumor to migrate to a new site. Urokinase is secreted by cancer cells and allows the cancer to invade the target organ and form secondary tumors. Urokinase converts plasminogen to the active plasmin which degrades protein in extracellular matrix, and plasmin also converts procollagenase to collagenase which degrades the collagen in membranes surrounding capillaries and lymph system.. Urokinase and streptokinase are also given after heart attacks to activate plasmin and reduce the chance of blood clot formation.