Distefano, M.D.

Bioorg. Med. Chem. Lett. 1999, 9, 79-84, 10.1016/S0960-894X(98)00684-2

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 wthin 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.

Distefano, M.D.

J. Am. Chem. Soc. 1997, 119, 11643-11652, 10.1021/JA970820K

In an effort to prepare selective and efficient catalysts for ester and amide hydrolysis, we are designing systems that position a coordinated metal ion within a defined protein cavity. Here, the preparation of a protein-1,10-phenanthroline conjugate and the hydrolytic chemistry catalyzed by this construct are described. Iodoacetamido-1,10-phenanthroline was used to modify a unique cysteine residue in ALBP (adipocyte lipid binding protein) to produce the conjugate ALBP-Phen. The resulting material was characterized by electrospray mass spectrometry, UV/vis and fluorescence spectroscopy, gel filtration chromatography, and thiol titration. The stability of ALBP-Phen was evaluated by guanidine hydrochloride denaturation experiments, and the ability of the conjugate to bind Cu(II) was demonstrated by fluorescence spectroscopy. ALBP-Phen-Cu(II) catalyzes the enantioselective hydrolysis of several unactivated amino acid esters under mild conditions (pH 6.1, 25 °C) at rates 32−280-fold above the background rate in buffered aqueous solution. In 24 h incubations 0.70 to 7.6 turnovers were observed with enantiomeric excesses ranging from 31% ee to 86% ee. ALBP-Phen-Cu(II) also promotes the hydrolysis of an aryl amide substrate under more vigorous conditions (pH 6.1, 37 °C) at a rate 1.6 × 104-fold above the background rate. The kinetics of this amide hydrolysis reaction fit the Michaelis−Menten relationship characteristic of enzymatic processes. The rate enhancements for ester and amide hydrolysis reported here are 102−103 lower than those observed for free Cu(II) but comparable to those previously reported for Cu(II) complexes.

Distefano, M.D.

Chem. Rev. 2001, 101, 3081-3112, 10.1021/cr000059o

Proteins are versatile molecules for catalyst design. Given the explosive development of molecular biology techniques for gene manipulation and functional selection, genetic approaches for enzyme design are currently in wide use. However, chemical methods provide a means for introducing a diverse range of functionality that does not occur in natural enzymes and cannot be easily incorporated by genetic methods. Thus, chemical modification of proteins remains a valuable tool for protein engineering. This article reviews a variety of approaches in which chemical modification has been used to alter the catalytic properties of existing enzymes to give new catalytic activities to existing enzymes or to impart catalytic activities to proteins normally devoid of enzymatic activity. Since the focus of this review is on the chemistry promoted by these systems, modifications that alter the overall stability or general physical properties of enzymes are not discussed here.