Stimulation and oxidative catalytic inactivation of thermolysin by copper•CoCys-Gly-His-Lys

[Cu2+center dot Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 mu M) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 mu M, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 mu M. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu2+-amino-terminal copper and nickel binding motif (lambda(max) similar to 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu2+ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (Delta E = 92 mV) for the Cu3+/2+ redox couple obtained for [Cu2+ center dot Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu2+ center dot Lys-Gly-His-Lys](+) (0.84 V, Delta E = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu2+center dot Lys-Gly-His-Lys](+) homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O-2) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k(obs) = 7.7 +/- 10(-2) min(-1), yielding a second-order rate constant of (7.7 +/- 0.9) x 10(4) M-1 min(-1). Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.