Function of the Carboxyl Group Specificity in Achromobacter Protease I

Abstract

Chemical modifications were made in the enzyme activity of Achromobacter protease I. Modification with carbodiimide in the presence or absence of the nucleophilic reagent ethylenediamine resulted in partial inactivation of enzyme activity. The rate of enzyme inactivation was reduced in the presence of butylamine, the most potent competitive inhibitor of the enzyme. Modification of the amino groups in the enzyme with acetic anhydride did not significantly change enzyme activity. These studies suggest that the specific feature of the enzyme was an essential carboxyl group. Because the alkylamines were competitive inhibitors of the enzyme, a homologous series of ω-aminoalkylagaroses were prepared with different lengths of hydrocarbon side chains (Seph-NH-(CH3₂)_n- NH₂, n=5_??_8). The affinity behavior of Achromobacter protease I towards these adsorbents was compared with that of trypsin. The enzyme was strongly adsorbed on adsorbents, as the hydrocarbon chain (n) increased from 5 to 8, while trypsin was not adsorbed on these adsorbents. Modification of either of the two amino acid residues (serine and histidine) with iPr₂P-F and Tos-LysCH₂C1 inactivated the catalytic activity and it simultaneously lost affinity to these adsorbents. The results indicate that a specific interaction between the amino cation at the tips of the hydrocarbon chains and the anion in the active site was concerned with the specific adsorption of the enzyme to ω-aminoalkylagarose. It was also found that the ω-aminoalkylagaroses were useful affinity adsorbents for the enzyme.