We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG(2)-3mD conjugate, but was dramatically reduced in the biotin-PEG(11)-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biofin-PEG(11), and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG(2)-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.