Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P1′,3 the residue furnishing the amino group to the scissile bond, produced significant changes in krmcat, whereas the identity of P1′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P1′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P1t,́ P2′, P3′) are involved while only two residues (P2, P1) on the amino terminal side of the sensitive bond are implicated.