Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-α convertase

We previously reported that tumor necrosis factor-alpha converting enzyme (TACE) was specifically inhibited by TIMP-3 but not TIMP-1, -2, and -4. Further mutagenesis studies showed that the N-terminal domain of TIMP-3 (N-TIMP-3) retained full inhibitory activity towards TACE. Full-length TIMP-3 and N-TIMP-3 exhibited indistinguishable values for the association rate constant and inhibitory affinity constant for the active catalytic domain of TACE (k(on) similar to 10(5) M-1 s(-1) and K-i(app) similar to0.20 nM). Moreover, their k(on) (similar to 10(4) M-1 s(-1)) and K-i(app) (similar to1.0 nM) values with a longer form of TACE (which encompasses the complete ectodomain including disintegrin, EGF and Crambin-like domains) were also shown to be similar. Detailed kinetic analyses indicated that TIMP-3 associated more quickly and with tighter final binding with TACE devoid of these C-terminal domains. We conclude that, unlike the interaction between many MMPs and TIMPs, the C-terminal domains of TIMP-3 and TACE are not essential in the formation of a tight binary complex. (C) 2001 Academic Press.