How Enzymes Work 11 Flashcards
Catalysis showers the activation energy for rxn
- 3 ways to increase rate of rxn: increase temp, increase conc of substances, add a catalyst
- catalysts lower the free energy of activation so that more reactions can occur relative to the uncatalyzed process
- catalysts bind and put the substrates in proximity, in the correct orientation, and provide functional groups for catalysis
- catalysts are regenerated during the reaction
- dont influence ratio of reactants to products
Enzymes are protein catalysts
-enzymes are the largest group of proteins
-have active sites that bind substrate/product and put substrates in proximity with the correct orientation and provide functional groups for catalysis
-enzymes preferentially bind and stabilize the transition state (induced fit)
Do not change the reaction equilibrium and are not chemically changed by the reaction
-are normally present in low amounts relative to the reactants and products
-enzymes enhance rates up to 10^17 fold
-carbonic anhydrase is a perfect catalyst
Cofactors are required by many enzymes
- some reaction require non-aminoacid cofactors: organic coenzymes or metal ions (apo vs halo-enzymes: without vs with cofactors)
- provide additional chemistries for carrying out chemical reactions
Cofactors: Metal ions or Coenzymes
Coenzymes: Cosubstrate or Prosthetic group
Cosubstrates: enter and exit the active site
Prosthetic groups: permanently bound
Enzyme catalytic mechanisms
- 3 types of catalysis used by enzymes:
- Acid-base catalysis: transfer or removal of H= lowers free energy of transition state
- Covalent catalysis: transient formation of a reversible covalent bond between enzyme and substrate
- Metal catalysis: direct or indirect role in catalysis: oxidation-reduction Rx
- amino acids side chains often play a direct role in acid-base and covalent catalysis at the enzyme active site
- enzymes have characteristic pH and temp optima
Nucleophiles and electrophiles in enzymes
- electron pair donor or negative charge can act as nucleophiles
- electron deficient atoms act as electrophiles
- nucleophiles attack electron-deficient centres to create a covalent bond
Metal ion catalysts
- eg. Alcohol dehydrogenase
- metal ions at the active site can mediate oxidation-reduction reactions, promote reactivity of other functionally groups, or interact directly with reacting substrate
- in alcohol dehydrogenase, a zinc ion stabilizes the developing O- during the transfer for a hydride (from coenzyme NADH) to the carbon
Example: chymotrypsin
- chymotrypsin is a member of the serine protease family with a preference for bulky non-polar residues on the carboxyl side of the peptide bond
- uses both covalent and acid-base catalysis in the hydrolysis of a peptide bond
- Asp 102, His 57, and Ser 195 form a conserved catalytic triad at the active site of Ser
- Asp 102 anchors His 57
- Ser195 acts as a nucleophile
- His 57 acts as a general base and later as a general acid
Chymotrypsin mechanism Step 1
- the peptide substrate enters the active site of chymotrypsin so that its scissile bond is close to the oxygen of Ser 195 (the N-terminal portion of the substrate is represented by Rn and the C-terminal Rc)
- removal of the Ser hydroxyl proton by His 57 (a base catalyst) allows the resulting nucleophile oxygen to attack the carbonyl carbon of the substrate
Chymotrypsin mechanism step 2
-the transition state, known as the tetrahedral intermediate, decomposes when His 57, now acting as an acid catalyst, donates a proton to the nitrogen of the scissile peptide bond. This step cleaves the bond. Asp 102 promotes the reaction by stabilizing His 57 through hydrogen bonding
Chymotrypsin mechanism step 3
-the departure of the C-terminal portion of the cleaves peptide, with a newly exposed N-terminus, leaves the N-terminal portion of the substrate (an acrylic group) linked to the enzyme. This relatively stable complex is known as the Cayley enzyme intermediate
Chymotrypsin mechanism step 4
-water then enters the active site. It donates a proton to His 57 (a base catalyst), leaving a hydroxyl group that attack the carbonyl group of the remains substrate. Similar to step 1
Chymotrypsin mechanism step 5
-in the second tetrahedral intermediate, His 57, now an acid catalyst, donates a proton to the Ser oxygen, leading to a collapse of the intermediate. This step is similar to step 2
Chymotrypsin mechanism step 6
-the N-terminal portion of the original substrate, now with a new C-terminus, diffuses away, regranting the enzyme
Chymotrypsin catalysis: other factors
- replacing Asp 102 or His 57 decreases activity by over 1000 fold, while replacing Ser 195 are at least 10^6 fold less active
- even with replacement of all 3 catalytic residues an enzyme can still enhance reaction by 10^5 fold due to proximity and orientation
- chymotrypsin is flexible, adopting slightly different conformations (induced fit) as it goes through the catalytic cycle
- preferentially stabilizes the transition state, through Gly 193 and a black one aside
Proteases
- other members of the serine protease family have similar tertiary structures (by evolution) but different substrate specificities foe to residues lining the substrate binding pocket
- some proteases (eg bacterial subtilisin) are not homologous with Ser proteases, but have similar mechanisms using a catalytic triad (convergent evolution)
- Isozymes: 2 different enzymes that catalyze the same reaction
