Chemical modification of porcine kidney aminopeptidase P indicates the involvement of two critical histidine residues

Abstract

Aminopeptidase P (AP-P), purified to homogeneity from porcine kidney membranes, was completely inactivated by treatment with 0.2 mM diethylpyrocarbonate (DEP) at pH 7.0. Treatment of the modified enzyme with 20 mM hydroxylamine resulted in recovery of AP-P activity. The differential absorption of native and modified AP-P at 240 nm showed that DEP modified two histidyl residues per mol of AP-P. The substrates, bradykinin(1-5) and Gly-Pro-Hyp, and also the inhibitor, apstatin, could protect against DEP inactivation. These results suggest that histidine residues are critical for AP-P activity.

The contribution of ionic interactions to the conformational stability and function of polygalacturonase from A. niger

“…The histidine residues in polygalacturonase could ionize in the pH range 5-7. Histidine residues have been implicated in the structure and conformational dynamics of proteins [15,16]. Chemical modification studies and an analysis of the kinetic parameters as a function of pH in polygalacturonase have indicated the presence of an essential histidine [17,18].…”

Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P)

“…X-prolyl aminopeptidase has been iden tified as a zinc metalloprotease, but does not contain any of the known zinc binding sites that classify the zinc metalloproteases in distinct families (Hooper, 1994). It has been shown by chem ical modification that two histidine residues of X-prolyl amino peptidase are essential for catalytic activity (Lim and Turner, 1996). Four conserved histidine residues were identified in the porcine sequence (H403.…”

Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P

“…The N-terminal signal sequence is underlined. Weight-matrix predictions [31] identify two possible cleavage points for the signal sequence, either at Gly 21 shown). There is a typical cleavable signal sequence at the Nterminus.…”

“…The present work represents the first example of the cloning of AP-P from a eukaryotic source. However, the sequence fails to reveal likely zinc-binding or catalytic residues, although chemical modification studies on the purified enzyme have indicated the presence of two critical histidine residues [21] whose location has yet to be determined. We are now in a position to initiate site-directed mutagenesis studies to identify these and other residues involved in the action of this new class of proteolytic enzyme.…”

“…Knowledge of the amino acid sequence of purified AP-P reveals that it does not possess the His-Glu-Xaa-Xaa-His motif typical of many zinc metallopeptidases, nor any other recognizable zinc-binding motif [20], although chemical modification studies have indicated that the protein contains two essential histidine residues [21]. The amino acid sequence of the mature protein [19] also, of course, lacks any N-terminal signal peptide that may be required for membrane targeting and the C-terminal signal sequence for co-ordinating the attachment of the GPIanchor moiety.…”