Kinetic properties of the α-lytic protease of Sorangium sp., a bacterial homologue of the pancreatopeptidases

The kinetics under consideration are those of a bacterial serine protease with the same "active serine" sequence as chymotrypsin, trypsin, and elastase, and with a single histidine residue in a sequence which closely matches the sequences around histidine-57 of chymotrypsin and the analogous histidine residues of trypsin and elastase. In agreement with previous evidence of an elastase-like specificity, esters of N-substituted, neutral, aliphatic L-amino acids proved to be good to excellent substrates for the α-enzyme; esters of arginine, tyrosine, and tryptophan were not hydrolyzed. The enzyme has a much higher activity than the pancreatopeptidases towards p-nitrophenyl acetate and p-nitrophenyl trimethyl acetate; the catalytic rate coefficient kc for the latter substrate is about fivefold greater than that of elastase.The catalytic properties match those of the pancreatopeptidases in the following respects. As demonstrated with N-acetyl-L-valine methyl ester as substrate, kc is dependent on an ionization with a pKa of 6.7 in water and 7.3 in H22O; Δ log (kc/Km)/ΔpH for this ionization is equal to 1.0; kc is reduced 50% when H2O is replaced by H22O. These findings are consistent with a requirement for a single unprotonated histidine residue and general basic catalysis by that residue. The burst of p-nitrophenol in hydrolyses of p-nitrophenyl trimethyl acetate is proportional to [E]0; the magnitude of the proportionality factor and the rate of attainment of a steady state are consistent with the condition [Formula: see text], as in chymotrypsin kinetics. Thus the purely catalytic properties of the α-enzyme match those of chymotrypsin very closely. These findings do not support reaction mechanisms which require two catalytically functional histidine residues for such catalysis. The substrate-binding properties of the α-enzyme differ from those of chymotrypsin in that substrate binding does not depend on ionization of an N-terminal α-amino group; Km for the hydrolysis of N-acetyl-L-valine methyl ester is constant from pH 5 to pH 10 and enzymatic activity is unaffected by acetylation of the enzyme's α- and ε-amino groups. Ks for the hydrolysis of p-nitrophenyl trimethyl acetate is appreciably greater than the Ks of elastase for this substrate.The chloromethyl ketones of glycine and valine did not inhibit the enzyme or alkylate its histidine residue.