Kinetics and Spectral Properties of Carboxypeptidase A Labeled with the 2-Hydroxy-5-nitrobenzyl Reporter Group

Abstract

Abstract Bovine carboxypeptidase A, labeled 1:1 with the hydroxynitrobenzyl reporter group, was isolated by gel filtration and triethylaminoethyl-cellulose chromatography following incubation of the enzyme with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium chloride in 20-fold molar excess at pH 7.5. Such labeling of carboxypeptidase occurred with the modification of a single mole of tryptophyl residues per mole as evidenced by amino acid analyses and fluorescence measurements. Comparison of the catalytic behavior of the hydroxynitrobenzylated enzyme with that of native carboxypeptidase A revealed a similar spectrum of activities toward aromatic ester, dipeptide, and tripeptide substrates, but somewhat modified kinetics. Examination of the spectral properties of the labeled enzyme revealed that the covalently bound hydroxynitrobenzyl chromophore is both optically active and sensitive to changes in its molecular environment. The chromophore appears to be situated in a generally hydrophobic environment within the enzyme molecule, not totally accessible to solvent. Interactions of the enzyme with substrates and inhibitors result in perturbations in both the absorption spectrum and the circular dichroic spectrum of the bound hydroxynitrobenzyl reporter group.