Acylation of thermolysin and of other neutral proteases with N-hydroxysuccinimide esters of acyl amino acid derivatives yields “superactive“ enzyme products that are more active than the native enzymes toward a variety of peptide substrates [Blumberg, S., & Vallee, B. L. (1975) Biochemistry 14, 2410]. Thermolysin modified with one such active ester, the N-hydroxysuccinimide ester of N-acetyl-p-(2, 4-dinitroanilino)-L-phenylalanine, was examined in detail to identify the site(s) of modification, responsible for the changes in activity. The chemical stability of the acyl-enzyme linkages indicates that tyrosine residues are largely modified. Although an average of about two residues can be modified by the reagent, evidence is provided that modification at only one distinct residue elicits the change in the catalytic properties. Fractionation of partially modified enzyme on an agarose-Gly3-d-Phe affinity column yields two protein fractions, one which is highly active and the other which is lower in activity. Digestion with thermolysin and separation of 2, 4-dinitro-phenyl-labeled peptides reveal that two major peptides composed of Ser, Glx, Gly, Tyr and Ser, Glx, Gly, Tyr2 constitute together about 70% of the labeled peptides in the highly active fraction, whereas in the fraction of lower activity their content is less than 20%. The N-terminal residues of these peptides are serine and tyrosine, respectively, and the C-terminal residues of both peptides are tyrosines. Since these analyses correspond uniquely to the overlapping sequences 107-110 (Ser-Gln-Gly-Tyr) and 106-110 (Tyr-Ser-Gln-Gly-Tyr) in the known sequence of thermolysin [Titani, K., et al. (1972) Nature (London), New Biol. 238, 35], it is concluded that modification of tyrosine-110 is responsible for superactivation of the enzyme. © 1979, American Chemical Society. All rights reserved.