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 x 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.