Artificial metalloenzymes based on protein cavities: Exploring the effect of altering the metal ligand attachment position by site directed mutagenesis

In an effort to construct catalysts with enzyme-like properties, we are emp loying a small, cavity-containing protein as a scaffold for the attachment of catalytic groups. In earlier work we demonstrated that a phenanthroline ligand could be introduced into the cavity of the protein ALBP and used to catalyze ester hydrolysis. To examine the effect of positioning the phenant hroline catalyst at different locations wthin the protein cavity, three new constucts - Phen60, Phen72 and Phen104 - were prepared. Each new conjugate was characterized by UV/vis spectroscopy, fluorescence spectroscopy, guani dine hydrochloride denaturation, gel filtration chromatography, and CD spec troscopy to confirm the preparation of the desired contruct. Analysis of re actions containing Ala-OiPr showed that Phen60 catalyzed ester hydrolysis w ith less selectivity than ALBP-Phen while Phen72 promoted this same reactio n with higher selectivity. Reactions with Tyr-OMe were catalyzed with highe r selectivity by Phen60 and more rapidly by Phen104. These results demonstr ate that both the rates and selectivities of hydrolysis reactions catalyzed by these constructs are dependent on the precise site of attachment of the metal ligand within the protein cavity. (C) 1998 Elsevier Science Ltd. All rights reserved.