Enzyme Mechanisms

ENZYMES: A. Enzyme Mechanisms 1.Proteases Introduction; roles and types Roles: Serine: Types: based on mechanism; Serine, Thiol, Carboxyl (acid), Metallo 2. Serine Proteases Reaction & specificity Activation (zymogens); Ile a-amino pKa somehow important Active Site Determination esterase activity burst kinetics => two-steps (Ping-pong ordered bi bi) protein modification pH studies X-ray crystallography Proposed mechanism 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 Chymotrypsin versus elastase 3. Other protease mechanisms Enzymes ENZYME MECHANISMS 1 Enzymes What is an Enzyme Mechanism? • In organic chemistry, there is a chemical mechanism. • In biochemistry, there is a chemical mechanism performed by the enzyme. • This mechanism may not have the same pathway as one in organic chemistry (think covalent catalysis). • An Enzyme Mechanism is a description of step-by-step what goes on at the active site, what are the intermediates, what order do they occur, and what are the rate constants (energetics and rate-limiting steps, etc.). Enzyme Mechanism Protease (Peptide hydrolase) • During digestion, dietary proteins must be broken down into small peptides by proteases. • During protein turnover in the cell, proteins must be broken down by proteases (lysosomal cathepsins, proteosome, etc.) • During regulation, protein processing: o Pro-collagen à collagen o Generation of endorphins o Blood clotting • During development, from fertilization (acrosome) onward 2 Enzyme Mechanism Protease (Peptide hydrolase) • Proteases are classified by their mechanism • Although there are hundreds of different proteases, there are only a few standard mechanisms that these proteins have converged on. 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 Enzyme Mechanism Protease (Peptide hydrolase) • Proteases are classified by their mechanism • Although there are hundreds of different proteases, there are only a few standard mechanisms that these proteins have converged on. 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 3 Enzyme Mechanism Serine Proteases N-term C-term Enzyme Mechanism Serine Proteases • Serine proteases are among the best studied enzymes. • Illustrate charge delocalization and transition-state stabilization by general acid/base catalysis • Also, illustrates how enzymes are regulated (for the first time for us)….. Zymogen activation. What is the reaction? This is a bi bi reaction. The specificity comes from R1 and R2 (mostly R1) 4 Enzyme Mechanism Chymotrypsin • This protease is able to cleave the peptide bond adjacent to aromatic amino acids. Chymotrypsin cuts this bond (the sessile bond). trypsin would not cut this peptide. ① ② ③ ④ OUTLINE • Regulation • Kinetic mechanism • Enzyme intermediates from protein modification studies • Enzyme intermediates from pH studies • Enzyme intermediates from structural studies • Enzyme mechanism and binding energy Enzyme Mechanism • Regulation Zymogens are Activated by Irreversible Covalent Modification Enteropeptidase is hormonally controlled in the duodenal mucosa S–S S–S 122 136 A B 201 C 5 Enzyme Mechanism Chymotrypsin • Regulation • This protease is activated by proteolysis, which leads to a conformational change. Active Site Active Site is buried Active Site is opened up • Kinetic mechanism Enzyme Mechanism Chymotrypsin • This protease cleaves peptide bonds with kcat value of ~50 s-1. • This is very fast and difficult to study • This protease cleaves ester bonds with slower kcat values (~0.1 s-1), but also the products (alcohols) are more easily measured using a spectrophotometer. What is the kinetic mechanism? • This is easier to study. Calculate Km & Vmax by measuring the rate of PNP formation versus [NAcPhe-PNPE], then at varying concentrations of water…..... wha wha NAcPhe-PNPE PNP 6 • Kinetic mechanism • Enzyme Mechanism Chymotrypsin React with p-nitrophenylacetate or N-acetyl-L-phenylalanine pnitrophenyl ester (NAcPhe-PNPE). • Monitor p-nitrophenolate (PNP) as a function of time • There are two rates: a fast “burst” rate and a slower steady-state rate. Ping-pong bi bi A Q (acid) F E E (E–Q = F) Acylation This is called pre-steady state kinetics B (water) P (ester) (alcohol) FAST Deacylation SLOW What is the nature of this enzyme intermediate (F)? • Protein modification studies • Enzyme Mechanism Chymotrypsin React with acylating reagent • Denature the enzyme; no reaction • Add substrate or inhibitor (b-phenylpropionate); reaction blocked/slowed • Use as an inhibitor of the whole class of Serine Proteases and b-phenylpropionate Esterases The nature of this enzyme intermediate (F) is an acylated enzyme at Ser-195 7 • Protein modification studies • Enzyme Mechanism Chymotrypsin React with Affinity Label TPCK • • Denature the enzyme; no reaction Add substrate or inhibitor; reaction blocked/slowed The enzyme must utilize a His-57 in the active site, near Ser-195. Does it activate the Ser (acting as a base)? • Enzyme Mechanism Chymotrypsin • pH studies Measure activity and enzyme constants as a function of pH 1) pH vs. kcat (Vmax) BH ← B: 2) pH vs. kcat/Km Something unprotonated is required in the ratelimiting step. Something protonated is required for binding. AH+ → A: Perhaps His-57 Perhaps an aminoterminal; Ile-16 Log kcat and kcat/Km gives a better measurement; RECALL: This is consistent with His-57 in the active site, acting as a base near Ser-195. 8 Enzyme Mechanism • pH studies From the book….. His-57 must be uncharged to perform at the active site (act as a base) Ile-16 at N-term must be + to keep active site open • structural studies Enzyme Mechanism Chymotrypsin • X-ray crystallography Chymotrypsin Elastase Trypsin In the active site, near Ser-195 & His-57, there is ALWAYS an Asp (Asp-102). This is called the “Catalytic Triad.” Ser-His-Asp 9 • structural studies • Enzyme Mechanism Chymotrypsin X-ray crystallography S–S Chymotrypsin Uses Two of our the Mechanistic Strategies S–S 42 58 122 136 S–S 201 168 182 191 S–S 220 S–S covalent acid/base Anion “Hole” Chymotrypsin Mechanism Step 1: Substrate Binding E S(A) ES (EA) Sessile bond 10 Chymotrypsin Mechanism Step 2: Nucleophilic Attack which hydroxyl EA F-A Chymotrypsin Mechanism Step 3: Substrate Cleavage F-A⇌F-QP P F-Q’ 11 Chymotrypsin Mechanism Step 4: Water Comes In F-Q’ What if His: were protonated to His:H+? Recall, the second half-reaction is slow B F-Q’•B Chymotrypsin Mechanism Step 5: Water Attacks F-Q F-Q’-B⇌F-Q F-Q’-B What if this His:H+ were deprotonated? 12 Chymotrypsin Mechanism Step 6: Break-off from the Enzyme E•Q F-Q Chymotrypsin Mechanism Step 7: Product Dissociates E Q E•Q 13 Chymotrypsin Mechanism Animation 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 14 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 15 • 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. In other words, pKa value of Asp slightly higher than that of Ser pKa values as amino acids are: The measured pKa values are: Asp102 – His57 – Ser195 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? 2.8 Å 2.0 Å 1.5 Å What is significant about LBHBs is that they are 4-5x stronger than normal 2.8 Å H-bonds (~20 kcal/mole) 16 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 Thr 226 Val 216 Ser 189 17 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 (Thiol) 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 18