Lecture 8: Enzyme Mechanisms

Enzyme Mechanisms

Enzyme catalyze reactions by lowering activation energy, mainly through stabilizing transition states. There are many ways how enzymes carry out catalysis and increase reaction rates.

• Tightest enzyme binding occurs at the transition state geometry, not the substrate.

Enzyme Reaction Mechanism: Proximity and Orientation Catalysis

- Enzyme

- Substrate

• Enzymes increate reaction rate by by binding to substrates in close proximity and aligning them in the correct orientation

• Reduces random tumbling of the substrate (entropy): makes reaction more likely to occur

• Substrates in correct positions give pseudo effect of increased substrate concentration

• pKa NOT changed

• Enzymes remove water from substrate, Desolvation involves the removal of tightly bound water from a substrate.

Enzyme Reaction Mechanism: Acid/Base Catalysis

- Enzyme

- Substrate

• Acid–base catalysis is when an enzyme lowers activation energy by protonating or deprotonating the substrate so bonds can break or form more readily during the reaction.

• An enzyme’s amino acid side chains can act as a proton donor (acid) and a proton acceptor (base) which makes bonds easier to break or form and this promotes rapid reaction

• If the enzyme donates a proton, it can make a bond easier to break, and if it removes a proton, it can make a molecule more reactive.

• Common amino acids involved: Histidine, Aspartate, Glutamine

• Histidine: Often involved because its R-group (imidaloze ring) has a pKA very close to neutral so can act as an acid or a base.

Enzyme Reaction Mechanism: Electrostatic Effects

- Enzyme: Oxyanion hole

- Substrate:

• Enzymes use charged amino acids in their active sites to help lower activation energy.

• Electrostatic destabilization involves an repulsion due to the like charges on the substrate that push the substrate away which causes it to change shape and go toward transition state geometry which reduces activation energy

• Oxyanion holes are enzymes that stabilize the unstable, negative oxyanion intermediates

Enzyme Reaction Mechanism: Covalent Catalysis

- Enzyme

- Substrate

• Enzyme forms a temporary covalent bond with the substrate

• Temporary intermediate makes the reaction easier to carry out

• Covalent catalysis is about making a bond, not about weak interactions like aromatic stacking, so does NOT involve stacking of aromatic R-groups

• Examples: Amino Acid Serine commonly forms temporary covalent bond, Cofactor Pyridoxal phosphate forms temporary covalent bonds during transamination reactions

Enzyme Reaction Mechanism: Chymotrypsin

- Enzyme: Chymotrypsin

- Substrate: Protein/peptide w Hydrophobic Amino Acid Chain

• Chymotrypsin is the enzyme and the substrate is a protein/peptide that contains a large hydrophobic aromatic amino acids(Phe,Tyr,Trp)

• Chymotrypsin recognizes proteins containing Phe,Tyr,Trp and cuts the peptide bond AFTER these amino acids

• The hydrophobic amino acid at the end (Ex.Phe) fits perfectly into the deep hydrophobic pocket in the active site of the Chymotrypsin enzyme

• Once the correct amino acid fits into the pocket, the substrate is held in the correct position, this aligns the peptide bond next to the aromatic amino acid for cleavage.

• Serine in the Chymotrypsin comes in the active site and cleaves the peptide bond.

• Two peptide fragments are released, and leave the active site

• is unchanged and ready to catalyze another reaction.

- Important amino acids in protein sequences that are R groups and are the active sites are not next to each other, the enzyme folds in a way that brings them together in 3D space.

- MANY different enzymes use serine to cut proteins, not just one original serine protease (an enzyme that breaks down peptide bonds using serine protease.

How does Serine do the cleaving in Chymotrypsin?

Catalytic Triad!! (AHS)

1. Aspartate stabilizes histidine, does this by having a negative charge that is ready to stabilize the positive charge it gains when it taking the H+ from serine.

2. Histidine activates serine, does this by taking one of serine’s hydrogen atoms, makes it reactive

3. Serine directly involved in the nucleophilic attack (meaning cleaving the peptide bond), does this by Ser-O- attacking the peptide carbonyl

4. Products formed: One fragment with an Amine end and one fragment with a Carboxylic Acid end.

5. After bond is broken the peptide fragment with the amine end leaves the enzyme first, the remaining forms into a temporary acyl enzyme intermediate.

- DIFP irreversibly inhibits chymotrypsin by covalently binding to the active-site serine (Ser195), permanently blocking the enzyme's catalytic activity by blocking serine to do any cleaving.

Enzyme Reaction Mechanism: Carbonic Anhydrase/Metal Ion Catalysis

- Enzyme: Carbonic Anhydrase

- Substrate: CO2

- Enzyme has a Zinc in its active site

1. Water enters the active site in an enzyme

2. Water is sitting near the zinc in the active site

3. Zinc weakens the water by making water loose an H+ so it becomes an OH-

4. OH- is super reactive, this is the reactive intermediate

5. OH- attacks CO2 (the substrate) and converts it into bicarbonate