Enzymes Regulation Lecture Note

ENZYMES: A. Enzyme Mechanisms 1. Proteases 2. Serine Proteases a. Reaction & specificity b. Activation (zymogens); Ile a-amino pKa somehow important c. Active Site Determination d. Proposed mechanism i. ii. iii. iv. Catalytic triad (Ser-His-Asp) – highly conserved Mechanism; tetrahedral intermediates and stabilize t.s. with oxy-anion hole Old; charge relay (Ser-195 pKa too high) Low-barrier Hydrogen bonds- Role for Asp e. Specificity i. Chymotrypsin versus elastase 3. Other protease mechanisms B. Enzyme Regulation 1. Introduction 2. Strategies a. Gene Regulation b. Covalent Modification c. Allosteric Control 3. Covalent Modification a. Proteolysis b. Protein modification i. Phosphorylation a) Kinases b) Phosphatase c) Example-PKA 4. Allosteric Control a.Regulation nomenclature Enzyme Regulation 1 Enzyme Regulation Recall that enzymes have 4 major attributes: 1. Increase rates of chemical reactions 2. Catalysis under mild conditions of temperature and pH 3. Very specific binding to substrates 4. Can regulate their activity Control of Enzyme Activity EXAMPLE: Enzyme Regulation Overview of Catabolism Interconnected and opposing pathways: CO2 e– ATP How are these opposing pathways regulated so that they are not working at the same time in a futile cycle? 2 EXAMPLE: Enzyme Regulation First: Catabolic & Anabolic Pathways Differ Enz-1 Enz-2 Enz-3 Enz-5 Enz-4 Second: Give an enzyme specific for each pathway an on/off switch EXAMPLE: Enzyme Regulation Enz-1 Enz-2 Enz-3 • This on/off switch is normally at the start of a pathway so all the intermediates need not be made; i.e., at the committal step. • This on/off switch is often controlled by a small molecule, often the product of the pathway; i.e., Negative Feedback Regulation 3 Enzyme Regulation So, how are cellular processes controlled? These controls are everywhere, from simple binding/release to embryological problems that set into motion all the processes to take a fertilized egg to an embryo. THERE ARE THREE BASIC STRATEGIES: 1. Genetic Control 2. Covalent-Modification Control 3. Allosteric Control Speed of each type of control: Slow (minutes) Medium (seconds) Fast (msec) Enzyme Regulation Control by Covalent Modification: Proteolytic activity Phosphorylation Adenylylation etc. Methylation 4 Enzyme Regulation Control by Covalent Modification: Phosphorylation ATP4- + R-OH ⇌ ADP3- + R-O-PO42- + H+ Kinase Now, if R = an enzyme g b a ATP4– ADP3– AMP2– Enzyme Regulation Control by Covalent Modification: ATP4- + Protein-OH ⇌ ADP3- + Protein-O-PO42- + H+ Protein Kinase What are the effects of introducing a phosphoryl group? What R-groups? Ser, Thr, Tyr Ionic environment H-bond acceptors DG = –6 kcal/mole Because enzymes are involved in this, there is a potential for amplification Protein– How stable is this modification? Kinetically stable; requires an enzyme 5 Enzyme Regulation Control by Covalent Modification: EXAMPLE: Protein Kinase A AKA: cAMPdependent Protein Kinase a2b2 (90 kDa & 40 kDa) a is Regulatory (R) b is Catalytic (C) and has the kinase activity The active site recognizes a particular site on substrate proteins Target sites have the consensus sequence: R RKXSB Inactive Active Large small hydrophobic Why is PKA inactivate when bound to R2? R has a sequence: RRGAI that fits into the C active site! Cyclic-AMP (cAMP) How is this equilibrium shifted? Enzyme Regulation Control by Covalent Modification: EXAMPLE: Protein Kinase A AKA: cAMPdependent Protein Kinase a2b2 (90 kDa & 40 kDa) a is Regulatory (R) b is Catalytic (C) and has the kinase activity The active site recognizes a particular site on substrate proteins Target sites have the consensus sequence: R RKXSB Inactive Active Large small hydrophobic Why is PKA inactivate when bound to R2? R has a sequence: RRGAI that fits into the C active site! Cyclic-AMP (cAMP) How is this equilibrium shifted? 6 Enzyme Regulation Control by Allostery: What is cAMP? H H2O phosphodiesterase For PKA, cAMP is known as a positive heterotropic allosteric effector Control by Allostery: Enzyme Regulation What is Allostery or Allosteric Control? (meaning: ‘allo’ = another, ‘steric’ = place, site, space) Control by binding to another site; NOT the active site Nomenclature for allosteric effector molecules: • molecules that are the substrate = homotropic • molecules that are the not the substrate = heterotropic • molecules that activate the enzyme = positive • molecules that inhibit the enzyme = negative Hence, cAMP is a positive heterotropic allosteric effector for PKA 7 Enzyme Regulation Control by Allostery: Recall binding curves: R+L KD = k1 k-1 Y RL [R][L] R+L [R • L] (Y) = Fraction bound (B) k1 k-1 RL [R][L] [R • L] K D = [R • L] (Y) [R]T (Y) [ L] Y= K D + [ L] Single kind of binding site 1.0 Fraction boundFraction (B) = [R • L] [R]T Y Bound 0.5 Multiple kinds of binding sites n [ L] n K D + [ L] D ` Y= Ligand concentration [L] WHY IS THIS SIGMOIDAL BEHAVIOR IMPORTANT FOR REGULATION? IF AFFORDS A WAY TO MAKE AN ON/OFF SWITCH Enzyme Regulation Control by Allostery: Example of famous binding proteins: 8 Control by Allostery: Enzyme Regulation Allosteric Effectors can change binding/kinetic behavior Non- allosteric enzyme (M-M enzyme) +positive allosteric effector Rate with M-M kinetics (+positive +positive effector effector) Rate with positive heterotropic allosteric effector (or som etim es these positive effectors convert the enzym e to a non-cooperative kinetics (M -M )). Rate with Sigmoidal kinetics Rate with negative heterotropic allosteric effector ON +negative allosteric effector OFF [S] If this is physiological concentration of substrate, we have an ON/OFF switch! From book: At increasing conc., positive effectors (blue) or negative effectors (red) can affect the Km or the Vmax to achieve the same switch. 9