Enzymes - The Most Important Class of Proteins on Earth

ENZYMES (The most important class of proteins on Earth) 1 Enzymes General Properties of Enzymes 1. 2. 3. 4. *t½ = 0.693 / kun(1 st order) kun = 0.693/t½ Enhance reaction rates Mild reaction conditions Reaction specificity Regulated activity Rate Enhancement by Enzymes * Enzymes General Properties of Enzymes 1. 2. 3. 4. *t½ = 0.693 / kun(1 st order) kun = 0.693/t½ Enhance reaction rates Mild reaction conditions Reaction specificity Regulated activity Rate Enhancement by Enzymes * 2 Enzymes Pioneers Buchner and his brother Hans (of the funnel fame) was first to separate biological catalysis (fermentation) from living cells by trying to make jam using yeast extracts as a preservative, which they called a zyme. Enzyme was derived from “in das zyme.” Sumner was the first to isolate and crystalize an enzyme, urease. It was all protein, therefore enzymes=protein Haldane was the first really theorize how enzymes worked. Enzymes You can write out the catalysis as a series of reactions: But, this is better understood as a CATALYTIC CYCLE E + S ⇌ ES ⇌ EP ⇌ E + P Divide the problem into two parts: Binding and Catalysis Enzyme•Substrate Complex Substrate (S) Binding Substrate is bound at the “active Enzyme site” (ES) Chemistry (E) Enzyme•Product Complex (EP) Product (P) • The rate that an individual enzyme can go around this cycle is called the “turnover number” • The turnover number is like a rate constant for catalysis, or kcat 3 Enzymes BINDING: Formation of the ES-complex Most Enzymes do not interact with their substrates like a lock and key. Rather, the enzyme changes shape upon substrate binding, a phenomenon called induced fit. 1852-1919 Conformational change Lock-and-key model Induced-fit model (proposed by Emil Fisher) (proposed by Dan Koshland) A hybrid model is called “ligand selective.” This says that many conformations are in equilibrium in solution and the substrate binds one best, pulling equilibrium. 1920-2007 Enzymes BINDING: First demonstration of Induced Fit Hexokinase: Glucose + ATP ⇌ Glucose 6-phosphate + ADP Hexokinase•Glc 6-P Hexokinase 4 Enzymes Hexokinase: Glucose + ATP ⇌ Glucose 6-phosphate + ADP Enzymes Enzymes bring substrates together to form an enzyme-substrate complex on a particular region of the enzyme called the active site. ES-complex The interaction of the enzyme and substrates at the active site promotes catalysis. The protein fold brings residues important for Binding & Catalysis to the Active Site. A schematic representation of the primary structure of lysozyme shows that the active site is composed of residues that come from different parts of the polypeptide chain. 5 Enzymes Enzyme-Substrate Complex Drives Selectivity Enzymes BINDING: great example of complementarity Example: Tyr-aminotransferase Weak interactions (non-covalent bonds) Hydrogen Bonds Salt bridges Van der Waals Hydrophobic effect This kind of complementarity is not restricted to ES complex: interactions of receptor-ligand, protein-protein, protein-nucleic acid, etc. Notice that this kind of complementarity allows for binding L-Tyr much better than D-Tyr. 6 Enzymes BINDING: Enzymes are stereo-selective …...even for molecules that are not chiral (so-called pro-chiral) Convert to Fisher projection Example: Lactate Dehydrogenase(LDH) Pro-S re-face (clockwise) H :H– C HO H3C H COO– C COO– L-Lactate (S) (counter clockwise) CH3 HO Pro-R :H– COO– HO C HO CH3 H COO– C si-face (counter clockwise) H CH3 D-Lactate (R) (clockwise) (:H–)(from sp2 The hydride NADH) can add to the carbonyl carbon from the top (on the re-face) or the bottom (si-face). But, it can only do ONE. Enzymes Binding affinities: examples • Binding of receptor and ligand • Receptors can be soluble or membrane-bound • Similar to binding of enzyme and substrate How do we measure the degree of the affinity? 7 Enzymes Receptor-Ligand binding A k1 R+L R+L A k-1 RL D k1 R R+ L k-1 k1 D k-1 KD = R+ LL Association reaction k1 k-1 reaction RDissociation L [R][L] [R • L] D A k1 = k-1 , etc. M What are the units of a dissociation constant? How about an association constant? M -1 How can we express how TIGHT a ligand binds to a protein? How can we measure KD? KD Enzymes: Receptor-Ligand binding Define fraction of R bound to a ligand = Y Define RT = total of all species of R Y =[R•L] / ([R]+[R•L]) Use Keq to calculate expression for free R KD = Substitute for free R RT = R + R L [ R • L] Y= [ R ]T [R][L] [R] = KD[R•L] / [L] [R • L] Y =[R•L] / (KD[R•L] / [L]+[R•L]) ÷[R•L] / [R•L] Y =1/(KD/[L] + 1) × [L] / [L] Y= [L] KD +[L] Equation for hyperbola à y = x/(b+x) Graph [L] vs fraction of receptor that is bound17 (Y) 8 10/6/21 Enzymes: Receptor-Ligand binding [ L] hyperbola à y = x/(b+x) Y= K D + [ L] (Y) = Fraction bound (B) Saturation binding [R • L] [R]T (Y) Hyperbolic Curve NOTE: what is Y when [L] = Kd? Kd Kd Enzymes: Receptor-Ligand binding Cooperative Binding: Multiple binding sites R+L k1 R R+ Ln k-1 k1 k-1 Multiple sites on R Y= R+ LnL n k1 R1.0L Still reaches saturation binding (Y) k-1 Fraction Bound 0.5 [ L] n K D + [ L] Better/easier binding D • Difficult/poor binding • Lag phase Ligand concentration [L] Sigmoidal Curve These curves (hyperbolic & sigmoidal) are called binding curves or “isotherms.” • Binding of a ligand at one site can affect the binding at other sites. 19 9 Enzymes Enzyme•Substrate Complex Substrate (S) (ES) Binding Enzyme (E) Chemistry Enzyme•Product Complex (EP) Product (P) Now that we have some concept of binding, the first important part of the catalytic cycle, lets discuss the second part of the cycle: Catalysis Four introductory aspects to Enzyme Catalysis: 1) 2) 3) 4) Rate enhancement How do you measure catalysis (enzyme activity) What is the relationship between reaction rate and [E]? Enzyme nomenclature a. reaction b. helpers c. enzymes Enzymes Catalysis 1) Rate Enhancement by Enzymes This ratio is sometimes called Proficiency TABLE 6-5 Some Rate Enhancements Produced by Enzymes Cyclophilin 105 Carbonic anhydrase 107 Triose phosphate isomerase 109 Carboxypeptidase A 1011 Phosphoglucomutase 1012 Succinyl-CoA transferase 1013 Urease 1014 Orotidine monophosphate (OMP) decarboxylase decarboxylase 1017 Uroporphyrinogen decarboxylase 2.5 × 1024 10 Enzymes Catalysis 2) How do you measure enzyme activity? S⇌P For this, you need an ASSAY. You can either measure the disappearance of S or the appearance of P (or couple to faster reactions that measure these indirectly; called a coupled assay) Rate (") = d[P]/dt Rate (") = – d[S]/dt Concentration (M) For example: LDH for pyruvate + NADH ⇌ D-lactate + NAD+ Which of these, S or P, can you measure? You can measure the loss of NADH or the appearance of NAD+ [P] Actually…. [S] slopes = rate (µM/min) Time (t) Enzyme activity has UNITS of µmoles P made, or S used, per minute U = µmole/min Enzymes More E Rate (!) = kcat D[S] Rate (!) = kcat[E] (µM•sec-1) (sec-1) (µM) More E Time (t) Rate (!) Time (t) [S] • The turnover number (kcat) is the number of catalytic cycles/unit time per enzyme active site. • Range of values from <1 to 107 sec-1 Concentration (M) [S] Concentration (M) 3) What is the relationship between reaction rate and [E]? Concentration (M) Catalysis [S] Time (t) No enzyme, “no rate” (really slow) Double the [enzyme], double the rate slope = 1.0 [E] Said to be “Diffusion Controlled” 11