But by the time of birth expression of the enzyme is repressed and chromosome breakage

522-12-3 structure apparent loss of activity that is associated with the last eluting inhibitor observed for wild-type lines, corresponding to unprocessed TI1. Given that no additional or later chromatographic peak having protease inhibitory activity was found in the mutant protein, it is likely that both forms of the TI1 protein co-eluted in peak 3. Analysis of seed protein extracts from the E109K mutant and corresponding wild-type lines on native gels confirms the apparent loss of the unprocessed TI1 protein due to the change in overall charge. Here both processed and unprocessed TI1 would be expected to be uncharged at pH 7.0. The impact of the mutations on the likely interaction between protease inhibitors and target enzymes was studied in terms of protein structure. Fig 6 shows the model of the wild-type TI1 in complex with trypsin, where the positions of the three mutations studied here are shown. The C77Y mutation, despite not being involved 681159-27-3 directly in the inhibitory domains, leads to a loss of one of the seven highly conserved disulphide bridges , and may be predicted from the model to lead to a loss of structural rigidity. In particular, this could adversely affect the presentation of the chymotrypsin inhibitory loop and therefore its efficacy as a substrate mimic. The S85F mutation affects the P1�� position of the inhibitory site that engages directly with the chymotrypsin active site and the substitution introduces a bulky aromatic side chain that would be predicted from the model to abrogate binding. In the case of the E109K, this region of the structure is not visible in any of the complexes that are available in databases , suggesting that it is flexible or cleaved and plays no significant role in the interaction between protease inhibitor and target enzyme. The position of E109 in Fig 6 is based on the structure of the free homodimeric inhibitor. However, it seems likely that E109 may be important in dimer formation, via an extended hydrogen- bonding network that would be important in such interactions. Although the E109K substitution may not disrupt these interactions, it could result in a different or disordered conformation for the carboxy-terminus and an over

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