SFP8+9 - Enzymes Flashcards
The active site is relatively small, how many amino acid residues may it be made up of and how many are directly involved in catalysis?
10-15 amino acid residues, 2-3 may be directly involved
What does the rest of the enzyme do?
Structural role
Enzymes use binding energy of substrate interaction to provide additional catalysis, true or false?
True
What enzyme is an example of only using bidning energy and no catalytic groups?
Tyrosyl tRNA synthetase
What is meant by binding energy?
Free energy released upon the interaction of a complementary enzyme and substrate
What is meant by the binding energy must stabilise the transition state of the reaction?
When the reactant binds to the catalyst, it forms a complex that is more stable than either the reactant or the catalyst alone. This binding energy can be used to stabilize the transition state and lower the activation energy of the reaction, increasing the rate of the reaction.
There is greater stabilisation at the enzyme-transition state complex than at the enzyme-substrate complex
The enzyme-transition state complex stabilises this high-energy intermediate by lowering its energy, making it easier for the reactants to proceed to the product
Upon binding, the substrate is distorted. Transition state makes better contacts than the substrate. Induced fit = structure of the enzyme is complementary to the TS only after binding and the active site closes around substrate to form a new environment for catalysis.
How does the enzyme bind to the substrate?
Large number of relatively weak non-covalent interactions
What type of bond to serine proteases hydrolyse?
Peptide bonds
Which carbon is attacked by serine protease?
Carbonyl
What is an example of a serine protease involved in digestion?
Chymotrypsin
What can be added to this to make it non-function (irreversible change)
Organophosphates
How does this work?
Organophosphate molecule binding covalently to the active site serine residue of the serine protease. The organophosphate forms a covalent bond with the serine hydroxyl group, resulting in the inactivation of the enzyme.
What is the triad of residues in serine proteases
Serine, Histidine, Aspartate
What are the frist two steps in the hydrolysis of a peptide bond by the catlytic triad?
- Substrate Binding: The substrate, typically a peptide or protein, binds to the active site of the serine protease. The active site consists of the catalytic triad residues, which are positioned in close proximity to the peptide bond to be cleaved.
- Activation of Serine: The histidine residue in the catalytic triad acts as a general base, abstracting a proton from the serine residue. This deprotonation of serine increases its nucleophilicity, allowing it to attack the carbonyl carbon of the peptide bond in the substrate.
3rd and 4th steps?
- Nucleophilic Attack: The activated serine residue, now in its nucleophilic form (serine-O⁻), attacks the carbonyl carbon of the peptide bond. This nucleophilic attack leads to the formation of a covalent acyl-enzyme intermediate, with the peptide bond temporarily attached to the serine residue.
- Histidine as General Acid: After the nucleophilic attack, the histidine residue acts as a general acid. It donates a proton to the leaving amino group, resulting in the cleavage of the peptide bond. This step generates a new N-terminus for the cleaved product.
How is water involved?
- Water Activation: A water molecule is now positioned for the next step. The aspartate residue in the catalytic triad helps to orient the water molecule and makes it more nucleophilic.
- Water Attack: The activated water molecule, now acting as a nucleophile, attacks the acyl-enzyme intermediate. This attack results in the breaking of the covalent bond between the serine residue and the substrate, restoring the serine residue to its original state.
What does the specificity of the of the serine proteases rely on? (for its targetting of the peptide bond)
The R-groups
Specific R groups for chymotrypsin?
Aromatic: Phe, Tyr, Trp
Specific R groups for Trypsin?
+ charged: Arg, Lys
Specific R groups for Elastase?
Small, hydrophobic: Ala
These are all endopeptidases. What is an endopeptidase?
They hydrolyse peptide bonds within the peptide chain
Where is the inactive form, chymotrypsinogen, produced?
Pancreas
When is it activated?
When the bond between Arg-15 and Ile-16 (Isoleucine) is cleaved by trypsin
Where is the developing negative charge from tetrahedral intermediates stabilised and by what ion?
Oxyanion hole, Mg2+
What happens to the binding affinity of restriction enzymes to their cognate sequences without Mg2+ being present?
Reduction in affinity to their specfiic sequences
What are isoenzymes/isozymes?
A group of enzymes that catlyse the same reaction but but have slightly different molecular structures
The amount of enzyme/protein present can be determined by protein expression or degradation. What are ways in which it can be expressed?
Signal to transcription factor, activate promoter, transcribe mRNA, process mRNA
What are ways in which the protein can be degraded?
Tag for degradation, direct to proteasome, degraded by proteasome
What is the rate of this like?
Slow and poor in responding to change. adaptation to long-term environmental changes
What are three regulatory strategies for fine control?
- Proteloytic activation
- Allosteric control
- Covalent modification (phosphorylation)
What is meant by proteolytic activation?
Enzyme is inactive until activated by cleavage of one or a few specific peptide bonds. E.g - chymotrypsinogen.
What is the pathway for the production of fibrin (required for blood clotting)
Prothrombin to thrombin. Cleaves fibrinogen to fibrin
What is meant by allosteric control?
Regulation of enzymatic activity through the binding of molecules to sites on the enzyme that are distinct from the active site. These regulatory molecules, known as allosteric effectors, can be either activators or inhibitors, and they modulate the enzyme’s catalytic activity by inducing conformational changes in the enzyme’s structure.
What type of inhibition is this?
Non-competitive inhibition
What is feedback inhibition?
A regulatory mechanism in which the final product of a metabolic pathway acts as an allosteric inhibitor of an enzyme earlier in the pathway.
What is covalent modification?
Attachment of regulatory groups to the enzyme via a covalent bond
What is the role of protein kinases?
Catalyse the transfer of phosphate groups to proteins
What is the donor molecule?
ATP
Where is the phosphate added to?
OH group
What does phosphatase do?
Removes a phosphate group
Why can the addition of a phosphoryl group cause a conformational change within a protein?
As it contains 2 negative charges and the potential for 3 hydrogen bonds
Which pathway are the enzymes which are often regulated by phopshorylation of dephosphorylation?
Metabolism
What do Cyclin-dependent Kinases (CdKs) control?
Timing of the cell cycle
