Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis - PubMed (original) (raw)

Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis

Jodie L Guy et al. FEBS J. 2005 Jul.

Abstract

Angiotensin-converting enzyme-2 (ACE2) may play an important role in cardiorenal disease and it has also been implicated as a cellular receptor for the severe acute respiratory syndrome (SARS) virus. The ACE2 active-site model and its crystal structure, which was solved recently, highlighted key differences between ACE2 and its counterpart angiotensin-converting enzyme (ACE), which are responsible for their differing substrate and inhibitor sensitivities. In this study the role of ACE2 active-site residues was explored by site-directed mutagenesis. Arg273 was found to be critical for substrate binding such that its replacement causes enzyme activity to be abolished. Although both His505 and His345 are involved in catalysis, it is His345 and not His505 that acts as the hydrogen bond donor/acceptor in the formation of the tetrahedral peptide intermediate. The difference in chloride sensitivity between ACE2 and ACE was investigated, and the absence of a second chloride-binding site (CL2) in ACE2 confirmed. Thus ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites. This is the first study to address the differences that exist between ACE2 and ACE at the molecular level. The results can be applied to future studies aimed at unravelling the role of ACE2, relative to ACE, in vivo.

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Figures

Figure 1

Figure 1

Schematic view of the active site of ACE2 and tACE. Binding interactions of the inhibitor (A) MLN‐4760 at the active site of ACE2 and (B) lisinopril at the active site of tACE. Hydrogen bonds to the ligand are shown (dotted lines). The different binding subsites are labelled. Adapted from [17].

Figure 2

Figure 2

Expression of soluble ACE2 mutants. Medium, taken from mock‐transfected (empty vector) HEK293 cells and HEK293 cells transiently expressing soluble ACE2, was concentrated in a 10‐kDa cut‐off column. Aliquots, containing 30 µg total protein, were separated by SDS/PAGE (6% polyacrylamide gel) and then analysed by immunoelectrophoretic blotting using a human ACE2 polyclonal antibody (top panel). Total protein (30 µg) was incubated with the ACE2‐specific fluorogenic peptide, Mca‐APK(Dnp) (25 µ

m

), as described in Experimental Procedures. Enzyme activity is expressed as mol product formed per min (bottom panel). Values are the mean of duplicate determinations.

Figure 3

Figure 3

Role of His505 and His345 in catalysis. Schematic of the proposed reaction intermediate of ACE2, showing the importance of His345 and His505. Hydrogen bonds to the ligand are shown (dotted lines).The equivalent residues in tACE are given in parentheses.

Figure 4

Figure 4

Effect of chloride ions on the activity of the ACE2 mutants (R169Q/R514Q). Medium, taken from HEK293 cells stably expressing soluble ACE2, was concentrated in a 10‐kDa cut‐off column and extensively dialysed against 50 m

m

Hepes/KOH buffer, pH 7.5, to remove chloride ions. Total protein (10 µg) was incubated with the ACE2‐specific fluorogenic peptide, Mca‐APK(Dnp) (25 µ

m

), as described in Experimental Procedures in the absence (grey) or presence (black) of NaCl (500 m

m

). Enzyme activity is expressed as mol product formed over time. Product was quantified using pure standards. Values are the mean of four independent determinations.

Figure 5

Figure 5

Activities of wild‐type and R169Q and R514Q ACE2 mutants in the absence (grey) and presence (black) of NaCl (500 m

m

). Medium, taken from HEK293 cells stably expressing soluble ACE2, was concentrated in a 10‐kDa cut‐off column and extensively dialysed against 50 m

m

Hepes/KOH buffer, pH 7.5, to remove chloride ions. Total protein (10 µg) was incubated with the ACE2‐specific fluorogenic peptide, Mca‐APK(Dnp) (25 µ

m

), as described in Experimental Procedures in the absence (grey bars) or presence (black bars) of NaCl (500 m

m

). Enzyme activity (mol product formed·min−1) is expressed as the percentage of activity with 500 m

m

NaCl. Product was quantified using pure standards. Values are mean ± SE from four independent determinations.

Figure 6

Figure 6

Chloride binding to ACE2 (yellow) and tACE (white). (A) Binding site of CL1 in ACE2 and tACE; (B) binding site of CL2 in ACE2 and tACE. Residue numbering for ACE2 is first. The chloride ion is green and the zinc ion is grey (both in spacefill). (B) The lisinopril ligand is coloured according to atom type (CPK) and the chloride ion is shown with a reduced radius to demonstrate its overlap with Glu398 in ACE2 more clearly.

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References

    1. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G & Turner AJ (2000) A human homolog of angiotensin‐converting enzyme: cloning and functional expression as a captopril‐insensitive carboxypeptidase. J Biol Chem 275, 33238–33243. - PubMed
    1. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R et al. (2000) A novel angiotensin‐converting enzyme‐related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res 87, E1–E9. - PubMed
    1. Hamming I, Timens W, Bulthuis MLC, Lely AT, Navis GJ & van Goor H (2004) Tissue distribution of ACE2 protein, the functional receptor for SARS cornavirus. A first step in understanding SARS pathogenesis. J Pathol 203, 631–637. - PMC - PubMed
    1. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F et al. (2002) Hydrolysis of biological peptides by human angiotensin‐converting enzyme‐related carboxypeptidase. J Biol Chem 277, 14838–14843. - PubMed
    1. Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira‐dos‐Santos AJ, da Costa J, Zhang L, Pei Y et al. (2002) Angiotensin‐converting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828. - PubMed

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