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

Ronald R. Davies, Hao Kuang, Dongfeng Qi, Aram Mazhary, Evelyn Mayaan, Mark D. Distefano

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24 Scopus citations

Abstract

In an effort to construct catalysts with enzyme-like properties, we are employing 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 phenanthroline catalyst at different locations within the protein cavity, three new constucts - Phen60, Phen72 and Phen104 - were prepared. Each new conjugate was characterized by UV/vis spectroscopy, fluorescence spectroscopy, guanidine hydrochloride denaturation, gel filtration chromatography, and CD spectroscopy to confirm the preparation of the desired contruct. Analysis of reactions containing Ala-OiPr showed that Phen60 catalyzed ester hydrolysis with less selectivity than ALBP-Phen while Phen72 promoted this same reaction with higher selectivity. Reactions with Tyr-OMe were catalyzed with higher selectivity by Phen60 and more rapidly by Phen104. These results demonstrate 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.

Original languageEnglish (US)
Pages (from-to)79-84
Number of pages6
JournalBioorganic and Medicinal Chemistry Letters
Volume9
Issue number1
DOIs
StatePublished - Jan 1999

Bibliographical note

Funding Information:
Acknowledgments. We thank Dr. C. Frieden and Dr. D. Bernlohr for providing the plasmids encoding the proteins used in this study. R. Davies was supported by a National Institutes of Health a'aining grant (NIH 2T32GM08347-06). This work was supported by a grant from the National Science Foundation (NSF-CHE-9506793).

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