Lecture 18 (10/27/17) Lecture 18 (10/27/17)

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1 Reading: Ch6; Lecture 18 (10/27/17) Problems: Ch5 (text); 2 Ch6 (study guide-facts); 5, 6, 7, 14 NEXT Reading: Ch5; 164, Problems: none Remember Monday at 6:30 in PHO-206 is the first MB lecture & quiz ENZYMES: A. Enzyme Mechanisms 1. Serine Proteases a. Proposed mechanism i. Catalytic triad (Ser-His-Asp) highly conserved ii. Mechanism; tetrahedral intermediates and stabilize t.s. with oxy-anion hole iii. Old; charge relay (but Ser-195 does not have the correct pka) iv. Low-barrier Hydrogen bonds- Role for Asp b. Specificity i. Chymotrypsin versus elastase 2. Other protease mechanisms B. 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 Lecture 18 (10/27/17) b) Phosphatase 1

2 Step 1: Substrate Binding Sessile bond Step 2: Nucleophilic Attack 2

3 Step 3: Substrate Cleavage Step 4: Water Comes In Recall, the second half-reaction is slow What if this were protonated to His:H +? 3

4 Step 5: Water Attacks What if this His:H + were deprotonated? Step 6: Break-off from the Enzyme 4

5 Step 7: Product Dissociates Animation 5

6 SUMMARY 1 st half-reaction 2 nd half-reaction Recall, the second halfreaction is slow E ❶ ❷ ❹ ❺ ❸ ❻ What evidence is in support of this mechanism? ❼ If you protonate the His-57, it can t get beyond the acylenzyme intermediate. Recall, the second half-reaction is slow. Perform X-ray crystallography but by trapping the enzyme in action! 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). sp 2 E=elastase (small aliphatic) sp3 S=YPFVEPI 6

7 The catalytic triad Enzyme mechanism and binding energy 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 pk a values should be similar. Asp102 His57 Ser195 pk a values as amino acids are: The measured pk a values are: (12.0)* 15.0 *only transiently Enzyme mechanism and binding energy 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) 7

8 Substrate Specificity Enzymes Chymotrypsin vs. Elastase Trypsin is much like Chymotrypsin except for a Asp-189 instead of Ser. Substrate Specificity Enzymes Chymotrypsin vs. Trypsin vs. Elastase 8

9 Enzymes Class of Protease Serine Thiol Acid Metal Examples Trypsin, Chymotrypsin, Elastase Papain, Cathepsin B, Caspases HIV protease, Pepsin, Cathepsin D, Renin, Chymosin Carboxylpeptidase A, Thermolysin Very similar with acylation/deacylation half-reactions Serine Protease Cysteine Protease Enzymes Class of Protease Serine Thiol Acid Metal Examples Trypsin, Chymotrypsin, Elastase Papain, Cathepsin B, Caspases HIV protease, Pepsin, Cathepsin D, Renin, Chymosin Carboxylpeptidase A, Thermolysin 9

10 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 1

11 EXAMPLE: 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? EXAMPLE: 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 2

12 EXAMPLE: 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 Another Example: Hemoglobin & Myoglobin in O 2 & CO 2 Transport Hb has high affinity = ON Hb has low affinity = OFF 3

13 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 a 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 Control by Covalent Modification: Proteolytic activity Phosphorylation Adenylylation etc. Methylation 4

14 Control by Covalent Modification: Phosphorylation ATP 4- + R-OH ADP 3- + R-O-PO H + Now, if R = an enzyme Kinase g b a ATP 4 ADP 3 AMP 2 Control by Covalent Modification: ATP 4- + Protein-OH ADP 3- + Protein-O-PO H + Protein Kinase What R-groups? Ser, Thr, Tyr What are the effects of introducing a phosphoryl group? Ionic environment H-bond acceptors DG = 6 kcal/mole Protein Because enzymes are involved in this, there is a potential for amplification How stable is this modification? Kinetically stable; requires an enzyme 5