hcAMPP constructs and protein expression.

<p><b>(A)</b> Wild-type and mutant hcAMPP orientation in the pET28a system employed in present study with the single point mutations at residue Y527 and R535, denoted as red and blue single-letter amino acid abbreviation, respectively. <b>(B)</b> hcAMPP wild-type, Y527F and R535A enzymes are shown in lane 1, 2 and 3, respectively, where M denoted the protein marker used in correspondence to show the molecular weight of each protein.</p

Steady-state kinetic parameters for wild-type and mutant hcAMPPs^a.

<p>Steady-state kinetic parameters for wild-type and mutant hcAMPPs<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#t001fn001" target="_blank">^a</a>.</p

Secondary structure analyses of wild-type and mutant hcAMPPs by circular dichorism.

<p>(<b>A</b>) CD spectra of wild-type, Y527F and R535A hcAMPPs are denoted by black, red and green circles, respectively. <b>(B)</b> CD spectra of wild-type (black line) and R535A (green line) hcAMPPs measured in the presence and absence of 10 mM guanidine hydrochloride. All spectra were measured at far-UV wavelengths ranging 250–190 nm and at a protein concentration of 0.35 mg/mL.</p

The effect of guanidine on the thermal stability of wild-type and R535A hcAMPPs monitored by circular dichorism.

<p>Temperature dependence of molar ellipticity at 222 nm measured for (<b>A</b>) wild-type hcAMPP and <b>(B)</b> R535A hcAMPP with (closed circle) or without (open circle) pre-incubation with guanidine hydrochloride.</p

Investigation of the proton relay system operative in human cystosolic aminopeptidase P

<div><p>Aminopeptidase P, a metalloprotease, targets Xaa-Proline peptides for cleavage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#pone.0190816.ref001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#pone.0190816.ref004" target="_blank">4</a>]. There are two forms of human AMPP, a membrane-bound form (hmAMPP) and a soluble cytosolic form (hcAMPP)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#pone.0190816.ref005" target="_blank">5</a>]. Similar to the angiotensin-I-converting enzyme, AMPP plays an important role in the catabolism of inflammatory and vasoactive peptides, known as kinins. The plasma kinin, bradykinin, was used as the substrate to conduct enzymatic activity analyses and to determine the Michaelis constant (<i>K</i>_<i>m</i>) of 174 μM and the catalytic rate constant (<i>k</i>_<i>cat</i>) of 10.8 s^-1 for hcAMPP. Significant differences were observed in the activities of Y527F and R535A hcAMPP mutants, which displayed a 6-fold and 13.5-fold for decrease in turnover rate, respectively. Guanidine hydrochloride restored the activity of R535A hcAMPP, increasing the k_cat/K_m 20-fold, yet it had no impact on the activities of the wild-type or Y527F mutant hcAMPPs. Activity restoration by guanidine derivatives followed the order guanidine hydrochloride >> methyl-guanidine > amino-guanidine > N-ethyl-guanidine. Overall, the results indicate the participation of R535 in the hydrogen bond network that forms a proton relay system. The quaternary structure of hcAMPP was determined by using analytical ultracentrifugation (AUC). The results show that alanine replacement of Arg535 destabilizes the hcAMPP dimer and that guanidine hydrochloride restores the native monomer-dimer equilibrium. It is proposed that Arg535 plays an important role in hcAMMP catalysis and in stabilization of the catalytically active dimeric state.</p></div

Kinetic analysis of MnCl₂ activation of wild-type, Y527F and R535A hcAMPPs.

<p><b>(A)</b> The rates of des-bradykinin (des-BK) product formation from bradykinin hydrolysis was plotted against the increasing concentrations of MnCl₂ and fitted with the Michaelis-Menten equation to determine the <i>Km</i> of co-factor on hcAMPP wild-type, (<b>B</b>) Y527 mutant and (<b>C</b>) R535A mutant enzymes.</p

Schematic diagram of hcAMPP proton transfer tunnel using PDB code 3CTZ.

<p>Domain I and II of hcAMPP are colored in light blue whereas the catalytic domain III is colored according to hydrophobicity. Positive and negative charged residues are color coded in red and blue, respectively whereas polar and hydrophobic residues are colored in grey. <b>(A)</b> The proton transfer tunnel (green dotted arrows) is embedded in hydrophobic surrounding formed by Tyr527, Arg535 and Asp415. Proton shuttling is managed through the hydrogen bonding at 2.8Å, between the side chain of Arg535 to η-hydroxyl of Tyr527 and γ-carboxylate of Asp415. <b>(B)</b> Surface charge representation of hcAMPP shown in comparison to <b>(A)</b>.</p

Sedimentation velocity profiles measured for wild-type and mutant hcAMPPs.

<p>The calculated hcAMPP <i>c(s)</i> distributions at the concentrations of 0.1 mg/mL (dotted line), 0.5 mg/ml (dashed line), and 1 mg/mL (solid line) for wild-type protein, Y527F mutant (red) and R535A mutant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#pone.0190816.ref064" target="_blank">64</a>] are shown on (<b>A</b>), (<b>B</b>) and (<b>C</b>), respectively. (<b>D</b>) The comparison of calculated <i>c(s)</i> distributions between 0.1 mg/mL proteins of wild-type (black triangle), Y527F (red circle) and R535A (green square) hcAMPPs as well as the effect of 10 mM guanidine hydrochloride supplementation on wild-type (black line) and R535A (green line) hcAMPPs are shown.</p

Thermal stability of wild-type and R535A hcAMPPs in the presence and absence guanidine hydrochloride (Gdn)^a.

<p>Thermal stability of wild-type and R535A hcAMPPs in the presence and absence guanidine hydrochloride (Gdn)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190816#t003fn001" target="_blank">^a</a>.</p