What is the currently accepted model for enzyme substrate binding?
Induced fit model
How many amino acids make up the active site and how many are directly involved in catalysis?
10-15 amino acids make up the active site but only 2-3 are directly involved in catalysis.
What are the five generalizations that can be made about all active sites?
- The active site is a 3D cleft or crevice.
- The active site takes up a small volume ~10-20% of the total enzyme.
- The active site is a unique microenvironment, normally excludes water.
- Substrates are bound to enzymes by multiple weak interactions. Non-covalent.
- The specificity of binding depends on the precisely defined arrangement of atoms in the active site.
Enzymes have an equilibrium constant of that ranges from…
…10^2 to 10^8 M corresponding to free energy interaction ranging from -13 to -50 kJ mol^-1.
Define the conformational selection hypothesis?
Suggests that enzymes exist in a variety of conformations, only some of which are capable of binding to a substrate.
What enzyme contains no catalytic groups and is solely catalyzed by binding energy?
Tyrosyl tRNA synthetase
Describe the induced fit model?
Enzyme binds the substrate via many relatively weak non-covalent interactions. In addition to chemical catalysis, binding energy between the enzyme and the substrate is used to stabilize the TS, lowering the activation energy, and increasing the rate of reaction.
What is the functions of chymotrypsin?
Protease
Which three residue are vital for catalytic function in chyotrypsin?
195-Serine, 57-Histidine, and 102-Aspartate
Describe the two step reaction in which chymotrypsin catalyses peptide bond cleavage?
- The acyl group of the substrate becomes covalently bound to the enzyme as the amide is released leaving an acyl-enzyme intermediate.
- The acyl-enzyme intermediate is hydrolysed to release the carboxylic acid component of the substrate and regenerating the free enzyme.
What is the role of 57-Histidine in chymotrypsin?
Positions the serine side chain and polarise its hydroxyl group so that it is poised for deprotonation. In the presence of the substrate His accepts the proton from the hydroxyl group of 195-serine, acting as a general base catalyst.
What is the role of 102-Aspartate in chymotrypsin?
Helps better orient the his residue and make it a better proton acceptor through hydrogen bonding and electrostatic effects.
Chymotrypsin, trypsin and elastase all catalyse specific peptide bond cleavages. What amino acids do each target?
Chromotrypsin = aromatic (bulky) amino acids e.g. Phe, Tyr, and Trp. Trypsin = positively charged e.g. Arg, Lys Elastase = small, hydrophobic e.g Ala.
Name the three regions within the active site of serine proteases?
- Activated serine site composed of the three amino acids.
- Oxyanion hole where the unstable intermediate is stabilised by Gly and Ser backbone residues. The H on these residues contributes a positive charge that stabilises the negative charge of the intermediate.
- Large hydrophobic pocket which is associated with specificity.
What is the name of the inactive form of chymotrypsin?
Chymotrypsinogen is produced in the pancreas.
How is the inactive form of chymotrypsin activated?
Peptide bond cleavage betwen Arg-15 and Ile-16 by trypsin. This cleavage results in the pi-chymotrypsin which cleaves other pi-chymotrypsin molecules to form alpha-chymotrypsin which is the fully active version.
What is the function of restriction endonucleases?
Catalyse the hydrolysis of phosphodiester backbone of DNA between 3’ oxygen and phosphorus.
Deascribe the allosteric regulation of aspartate transcarbamoylase
Catalyses the first step in the biosynthesis of pyridines; the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-aspartate and orthophosphate.
Final product cystidine triphosphate (CTP) inhibits asapartate transcarbamolyase.
Allosterically regulated enzymes do not produce the typical M-M curve instead produce a…
…sigmoidal curve.
What are the two isoforms of lactate dehydrogenase and how do they differ?
H form in the heart has a higher affinity for substrates and high levels of pyruvate inhibit H.
L form in the liver has a weaker affinity for substrates and is not inhibited by high levels of pyruvate.
How does phosphorylation effect protein structure?
Phosphoryl group adds two negative charges and the potential for three H bonds which can result in a confirmational changeor formation of a new binding site.
Which amino acids typically under phosphorylation?
Ser/Thr or Tyr.
Which amino acid is typically aceylated?
Lysine
How does aceylation effect histone?
More acetyl groups = more transcription
