Enzyme Mechanisms Lecture Note

ENZYMES: A. Enzyme Mechanisms 1. Serine Proteases a. Proposed mechanism i. ii. iii. iv. b. Catalytic triad (Ser-His-Asp) – highly conserved Mechanism; tetrahedral intermediates and stabilize t.s. with oxy-anion hole Old; charge relay (but Ser-195 does not have the correct pKa) Low-barrier Hydrogen bonds- Role for Asp Specificity i. Chymotrypsin versus elastase 2. Other protease mechanisms B. Enzyme Regulation 1. Introduction 2. Strategies a. Gene Regulation b. Covalent Modification c. Allosteric Control 3. Covalent Modification 1. Proteolysis 2. Protein modification i. Phosphorylation a) Kinases b) Phosphatase 1 SUMMARY Recall, the second halfreaction is slow 1st half-reaction 2nd half-reaction ❶ ❷ ❺ E ❹ ❸ What evidence is in support of this mechanism? ❻ ❼ Chymotrypsin Mechanism Recall, the second half-reaction is slow. Perform X-ray crystallography but by trapping the enzyme in action! If you protonate the His-57, it can’t get beyond the acylenzyme intermediate. sp2 E=elastase (small aliphatic) If you de-protonate during turnover and stop the reaction, its built up at the slowest step (the His-57 proton is titrated and can’t participate in breakdown of the tetrahedral intermediate). sp3 S=YPFVEPI 2 • Enzyme mechanism and binding energy Chymotrypsin Mechanism The “catalytic triad” Asp102 – His57 – Ser195 The catalytic triad is found in all Serine Protease and Serine Esterases (e.g., acetylcholinesterase) How does this work? For this to work, all pKa values should be similar. Asp102 – His57 – Ser195 pKa values as amino acids are: The measured pKa values are: 3.5 7.0 6.0 15.0 7.0(12.0)* 15.0 *only transiently • Enzyme mechanism and binding energy Chymotrypsin Mechanism Low-barrier H-bond in the Transition State? What is a Low-Barrier H-bond? Where is the LBHB in the triad? What is significant about LBHBs is that they are 4-5x stronger than normal 2.8 Å H-bonds (~20 kcal/mole) 3 Enzymes Substrate Specificity Chymotrypsin vs. Elastase Trypsin is much like Chymotrypsin except for a Asp-189 instead of Ser-189. Enzymes Substrate Specificity Chymotrypsin vs. Trypsin vs. Elastase 4 Enzymes Class of Protease Examples Serine Trypsin, Chymotrypsin, Elastase Thiol Papain, Cathepsin B, Caspases Acid HIV protease, Pepsin, Cathepsin D, Renin, Chymosin Metal Carboxylpeptidase A, Thermolysin Very similar with acylation/deacylation half-reactions Serine Protease Cysteine Protease Enzymes Class of Protease Examples Serine Trypsin, Chymotrypsin, Elastase Thiol Papain, Cathepsin B, Caspases Acid HIV protease, Pepsin, Cathepsin D, Renin, Chymosin Metal Carboxylpeptidase A, Thermolysin 5 Enzyme Regulation 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 6 EXAMPLE: Enzyme Regulation Overview of Catabolism Interconnected and opposing pathways: CO2 e– ATP EXAMPLE: How are these opposing pathways regulated so that they are not working at the same time in a futile cycle? 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 7 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 Enzyme Regulation Another Example: Hemoglobin & Myoglobin in O2 & CO2 Transport Hb has high affinity = ON Hb has low affinity = OFF 8 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) *NOTE: The VV&P textbook (Chapter 14) lists 4 strategies, but one called “substrate Enzyme Regulation Control by Covalent Modification: Proteolytic activity Phosphorylation Adenylylation etc. Methylation 9 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 10 Enzyme Regulation Control by Covalent Modification: EXAMPLE: Protein Kinase A 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 hydrophobic small 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 Allostery: What is cAMP? H H2O phosphodiesterase For PKA, cAMP is known as a positive heterotropic allosteric effector 11