Enzymes - Harmer 3 Flashcards
Knowing enzyme function and structure (of the active site particularly) in molecular detail is important because
some active sites can be targeted by drugs, and stop the organism from evolving around the drug
some proteins are unique to viruses and bacteria, this makes them targets
e.g. HIV protease binds the drug Nefinavir in its active site which inhibits the enzyme
Chymotrypsin is a
serine protease
serine proteases have a catalytic serine residue in the active site that plays a nucleophilic role
Chymotrypsin info
chymotrypsin cleaves the peptide bond on the carboxyl side of bulky and hydrophobic amino acids
chymotrypsin uses COVALENT catalysis: the serine residue acts as a powerful nucleophile that forms a temporary covalent bond with the substrate
serine 195 is the main component of the active site
chymostrypsin uses a 2 step process to hydrolyse the peptide bond (forms an acyl-enzyme intermediate) then a water molecule hydrolyses the bond to form a hydrolysed product and regenerated OH on serine
Chymostrypsin chemistry
- the first step is a fast acylation step in which the acyl group of the substrate is added on to the enzyme and an amide is released
- the second step is slower and involves the hydrolysis of the intermediate complex with water as a nucleophile
The catalytic triad of chymotrypsin
contains apartate (102), histidine (57) and serine (195)
the serine residue acts as a nucleophile and attacks the carbonyl of the substrate
the side chain of serine exists in its alcohol form OH, which is not a good nucleophile
aspartate and histidine transform the alcohol into O-, the conjugate base, which is a strong nucleophile. They do this by: aspartate O- interacts with the (delta +) on histidine to form a hydrogen bond. This repositions the other side chain of histidine in a favourable conformation to allow the nitrogen on histidine to pull the H away from the O on serine, creating the nucleophile.
Chymotrypsin mechanism
aspartate repositions histidine so its electrons can interact with serine OH and create O-, the nucleophile.
O- attacks the carbon on the carbonyl group, forming a tetrahedral intermediate. This is stabilised by the oxyanion pore (N-Hs inside the pocket interact with the O- of the intermediate and stabilise it). The intermediate quickly collapses and the lone pair on O form a pi bond with carbon and cleaves the molecule forming an amide on histidine and an acyl on serine.
the amide pulls the H of histidine and departs creating room in the active site for a water molecule to hydrolyse the peptide bond. Histidine pulls an H off H2O, creating the nucleophile OH-. OH- attacks the carbon of the acyl attached to serine, forming again an unstable tetrahedral intermediate which collapses and forms a carboxylic acid product and regenerates serine.
Hydrophobic pockets
allow specificity
substrates often contain hydrophobic areas and these can be used to increase affinity or distinguish between substrates
the enzyme produces a hydrophobic region complementary to the substrate - other substrates will have either a steric clash, an incorrect H bonding pattern, or leave a water filled hole.
e.g. chymotrypsin S1 pocket is deep, long and hydrophobic, binds preferentially to a tyrosine residue which fits in the hydrophobic pocket
Main chain stabilisation
peptide bonds are polarised: in a peptide, the NH part is delta + and the CO is delta-
these groups can be stacked together in a folded protein to create slightly positive areas and slightly negative areas
charge-charge interactions of these areas and the substrate reduce the Ea and make the TS more favourable
Co localising substrates
form near attack complexes that significantly increase the reaction rate
the enzyme holds 2 substrates both near each other and in the right conformation so that the reactive parts are presented and increases the chances of a correct collision
