Serine Protease Lab Report

• Serine, threonine and cysteine proteases use a nucleophilic residue (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme.

A comparison of the two hydrolytic mechanisms used for proteolysis. enzyme is shown in black, substrate protein in red and water in blue.The top panel shows 1-step hydrolysis where the enzyme uses an acid to polarise water which then hydrolyses the substrate. The bottom panel shows 2-step hydrolysis where a residue within the

The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all superfamilies of serine protease enzymes. The triad is a coordinated structure consisting of three amino acids: His 57, Ser 195 (hence the name "serine protease") and Asp 102. These three key amino acids each play an essential role in the cleaving ability of the proteases. While the amino acid members of the triad are located far from one another on the sequence of the protein, due to folding, they will be very close to one another in the heart of the enzyme. The particular geometry of the triad members are highly characteristic to their specific function: it was shown that the position of just four points of the triad characterize the function of the containing

The next step is nucleophilic attack by the deprotonated cysteine's anionic sulfur on the substrate carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. Therefore, they are also sometimes referred to as thiol proteases. The thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate fragment, while regenerating the free enzyme.

3.Mechanism of threonine protease

Threonine proteases use the secondary alcohol of their N-terminal threonine as a nucleophile to perform catalysis.( Brannigan,etal1995) The threonine must be N-terminal since the terminal amine of the same residue acts as a general base by polarising an ordered water which deprotonates the alcohol to increase its reactivity as a nucleophile.( Ekici, OD 2008)

Catalysis takes place in two