Chemical Modification of Amino Groups in Mucor miehei Aspartyl Proteinase, Porcine Pepsin, and Chymosin. I. Structure and Function

Abstract

The effects of chemical modification of ɛ-amino groups (using the water soluble polyanionic copolymer ethylene/maleic anhydride) on the structure and function of various aspartyl proteinases (the proteinase from the fungus Mucor miehei (MMP), and the mammalian proteinases porcine pepsin and chymosin) were examined. Modification of 100% of the lysyl residues in the proteinases resulted in an increased milk-clotting to proteolytic activity ratio (MC/PA) for pepsin and chymosin, and a lower MC/PA for MMP. Amino-modified MMP was insensitive to the proteinase inhibitor pepstatin, but modified pepsin and chymosin were inactivated. Modification shifted the pH-activity optimum of the mammalian enzymes by 0.5 pH units to a more alkaline pH and to a more acidic optimum for MMP. Amino modification also decreased the thermostability of the fungal enzyme. Changes in tertiary structure and significant differences in the proportions of the secondary structure fractions α-helix and β-sheet were evident only for modified MMP. Results from this study suggested that MMP was more susceptible to destabilization by charge alteration than aspartyl proteinases of mammalian origin. © 1991, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.