An inhibitory monoclonal antibody binds in close proximity to a determinant for substrate binding in cytochrome P450IIC5.

摘要: We used the expression of chimeric proteins and point mutants to identify amino acids hepatic progesterone 21-hydroxylase P450IIC5 which are part an epitope recognized by inhibitory monoclonal antibody affect substrate binding. Three at positions 113, 115, 118 were introduced into P450IIC4, is 95% identical P450IIC5. The resultant protein acquired binding 1F11, highly specific for Point in P450IIC4 showed that two three changes, T115S N118K, contribute this antibody. mutant bound weakly (Kd greater than 30 nM) whereas N118K as tightly less or equal 0.7 nM). Thus, residues 115 located on surface these enzymes, Lys/Asn difference acid largely responsible high degree discrimination exhibits between P450IIC4. valine alanine mutation position 113 conferred a lower apparent Km 21-hydroxylation. Because was not affected mutation, it tempting speculate residue buried where exerts its effect catalytic activity interaction with alters active site. close proximity Ala113 suggests interferes