THE HEMOREGULATORY PEPTIDE N-ACETYL-SER-ASP-LYS-PRO IS A NATURAL AND SPECIFIC SUBSTRATE OF THE N-TERMINAL ACTIVE-SITE OF HUMAN ANGIOTENSIN-CONVERTING ENZYME

Angiotensin I-converting enzyme (ACE) is a zinc-dipeptidyl carboxypept idase, which contains two similar domains, each possessing a functiona l active site, Respective involvement of each active site in the degra dation of the circulating peptide N-acetyl-seryl-aspartyl-lysyl-prolin e (AcSDKP), a negative regulator of hematopoietic stem cell proliferat ion, was studied by using wild-type recombinant ACE and two full-lengt h mutants containing a single functional site, Both the N- and C-activ e sites of ACE exhibit dipeptidyl activity toward AcSDKP, with K-m val ues of 31 and 39 mu M, respectively, However, the N-active site hydrol yzes the peptide 50 times faster compared with the C-active site, with k(cat)/K-m values of 0.5 and 0.01 mu M(-1).s(-1), respectively, The p redominant role of the N-active site in AcSDKP hydrolysis was confirme d by the inhibition of hydrolysis using a monoclonal antibody specific ally directed against the N-active site, The N-domain specificity for AcSDKP will aid the identification of specific inhibitors for this dom ain. This is the first report of a highly specific substrate for the N -active site of ACE, with kinetic constants in the range of physiologi cal substrates, suggesting that ACE might be involved via its N-termin al active site in the in vivo regulation of the local concentration of this hemoregulatory peptide.