Flap cleavage

Nuclease cleavage (flap cleavage, 5 -nuclease cleavage, or restriction enzyme cleavage). Minisequencing or primer extension. 

In conclusion, the double mutant pepsin T77D/G78(S)S79 was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contribute to the Si substrate specificity for basic amino acid residues in aspartic proteinases. 

The protease exists as a homodimer. Each 99-residue monomer contains 10 j3-strands and the dimer is stabilized by a four-stranded antiparallel jS-sheet formed by the N- and C-terminal strands of each monomer. The active site of the enzyme is formed at the interface, where each monomer contributes a catalytic triad (Asp2 -Thr2 -Gly ) that is responsible for cleavage of the protease substrates. The "flap region" is located above the reactive site and is formed by a hairpin from each monomer of two antiparallel j3-strands joined by a j8-turn. There is little difference between the solution and crystal structures of protease-inhibitor complexes, except in those regions where the polypeptide chain is disordered. However, experiments in solution have allowed access to parameters that are not directly accessible from crystal data. These parameters, such as the amplitude and frequency of backbone dynamics, the protonation states of the catalytic aspartate residues, and the rate of monomer interchange, are essential in understanding the interaction of HIV protease with potent inhibitors. 

Aspartic proteases form a group of proteolytic enzymes that catalyze peptide bond cleavage by acid-base catalysis and activation of a water molecule for nucleophilic attack on the amide carbon. Crystal structures of mammalian and fungal enzymes are known. In pepsin, the best studied aspartic protease, catalysis proceeds by water activation and leaving group protonation. Both involve an aspartate, which explains the low pH optimum of around 4. The two aspartic acid residues are situated around a hydrophobic deft that can accommodate seven amino acids (the S4-S3 subsites). The active site is covered by a flexible flap, which contributes to SI subsite specificity. 

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