Arg1098 Is Critical for the Chloride Dependence of Human Angiotensin I-converting Enzyme C-domain Catalytic Activity

Liu, Xifu; Fernandez, Marian; Wouters, Merridee A.; Heyberger, Sophie; Husain, Ahsan

doi:10.1074/jbc.m101495200

Cited by 57 publications

(85 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both ACE activity, and inhibitor affinity for ACE have previously been shown to be dependent on chloride ions, with cACE exhibiting this to a greater extent than nACE 8, 21, 22, 23, 24. As observed with previous structures, all the complexes described here contain the usual two chloride ions bound to cACE, and one to nACE.…”

Section: Resultsmentioning

confidence: 97%

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

et al. 2018

View full text Add to dashboard Cite

Angiotensin‐1‐converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (known as nACE and cACE) with different substrate specificities. Based on kinetic studies it was previously reported that sampatrilat, a tight‐binding inhibitor of ACE, K i = 13.8 nm and 171.9 nm for cACE and nACE respectively [Sharma et al., Journal of Chemical Information and Modeling (2016), 56, 2486–2494], was 12.4‐fold more selective for cACE. In addition, samAsp, in which an aspartate group replaces the sampatrilat lysine, was found to be a nonspecific and lower micromolar affinity inhibitor. Here, we report a detailed three‐dimensional structural analysis of sampatrilat and samAsp binding to ACE using high‐resolution crystal structures elucidated by X‐ray crystallography, which provides a molecular basis for differences in inhibitor affinity and selectivity for nACE and cACE. The structures show that the specificity of sampatrilat can be explained by increased hydrophobic interactions and a H‐bond from Glu403 of cACE with the lysine side chain of sampatrilat that are not observed in nACE. In addition, the structures clearly show a significantly greater number of hydrophilic and hydrophobic interactions with sampatrilat compared to samAsp in both cACE and nACE consistent with the difference in affinities. Our findings provide new experimental insights into ligand binding at the active site pockets that are important for the design of highly specific domain selective inhibitors of ACE.DatabaseThe atomic coordinates and structure factors for N‐ and C‐domains of ACE bound to sampatrilat and sampatrilat‐Asp complexes (6F9V, 6F9R, 6F9T and 6F9U respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

show abstract

Section: Resultsmentioning

confidence: 97%

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

et al. 2018

View full text Add to dashboard Cite

show abstract

“…On the other hand, site-directed mutagenesis studies suggest that the Cl Ϫ -coordinating ligand Arg 1098 of sACE (Arg 522 of tACE) at the second chloride ion site in tACE (CL2) plays a role in anion activation in sACE (47). The equivalent residue in ACE2 (Arg 514 ) cannot play the same role because this chloride-binding site is replaced in ACE2 with the Glu 398 / Ser 511 double substitution.…”

Section: Resultsmentioning

confidence: 99%

ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

Towler

Staker²,

Prasad³

et al. 2004

Journal of Biological Chemistry

663

841

View full text Add to dashboard Cite

The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ؉ E zincbinding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2-and 3.0-Å resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (ϳ16°) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino}-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S 2 substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.The angiotensin-converting enzyme (ACE) 1 -related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ϩ E zinc-binding consensus sequence (1, 2). The catalytic domain of ACE2 is 42% identical to that of its closest homolog, somatic angiotensinconverting enzyme (sACE; EC 3.4.15.1), a peptidyl dipeptidase that plays an important role in the renin angiotensin system for blood pressure homeostasis. The loss of ACE2 in knockout mice has no effect on blood pressure, but reveals ACE2 as an essential regulator of heart function (3). In a recent discovery, ACE2 was identified as a functional receptor for the coronavirus that is linked to the severe acute respiratory syndrome (SARS) (4, 5).The physiological differences observed in the phenotypes of ACE (6, 7) and/or ACE2 (3) knockout mice presumably reflect the significant differences in substrate specificity and reactivity between these enzymes. Many substrates for ACE2 were identified by screening biologically active peptides (8). In all cases, only carboxypeptidase activity was found. Of the seven best in vitro peptide substrates identified (k cat /K m Ͼ 10 5 M Ϫ1 s Ϫ1 ), proline and leucine are the preferred P 1 residues, with a partiality for hydrophobic residues in the P 1 Ј position, although basic residues at P 1 Ј are also cleaved (peptide-binding subsites in proteins are as previously defined (9)). Some of the best in vitro peptide substrates are apelin-13, des-Arg 9 -bradykinin, angiotensin II, and dynorphin A-(1-13). The longest peptide substrate identified is a 36-residue peptide, apelin-36 (8). An examination of the ACE2 and ACE literature may be found in recently published reviews (10 -12).We report here the first crystal ...

show abstract

“…The chloride dependence of ACE has long been recognized [25], and most recently mutagenesis studies have shown that it is in fact an arginine residue (Arg1098) that is essential for the chloride activation of ACE [18]. The structure of tACE revealed the location of two buried chloride ions [19].…”

Section: Comparing the Chloride-binding Sites Of Tace And Ace2mentioning

confidence: 99%

“…Most recently, ACE2 has been identified as a functional receptor for the coronavirus which causes the severe acute respiratory syndrome (SARS) [14]. For recent reviews, see [15,16].ACE2 shares a number of characteristics with ACE, both being zinc-containing enzymes which are sensitive to anion activation [4,17,18]. However, unlike ACE, ACE2 functions as a carboxypeptidase and is not susceptible to inhibition by the classical ACE inhibitors [1,2].…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Guy¹,

Jackson²,

Jensen³

et al. 2005

The FEBS Journal

119

125

View full text Add to dashboard Cite

Angiotensin-converting enzyme-2 (ACE2) is a membrane protein with its active site exposed to the extracellular surface of endothelial cells, the renal tubular epithelium and also the epithelia of the lung and the small intestine [1][2][3]. Here ACE2 is poised to metabolize circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by angiotensin-converting enzyme (ACE; EC 3.4.15.1) [1,4]. Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the concentrations of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7) [5][6][7][8][9][10][11][12][13]. Most recently, ACE2 has been identified as a functional receptor for the coronavirus which causes the severe acute respiratory syndrome (SARS) [14]. For recent reviews, see [15,16].ACE2 shares a number of characteristics with ACE, both being zinc-containing enzymes which are sensitive to anion activation [4,17,18]. However, unlike ACE, ACE2 functions as a carboxypeptidase and is not susceptible to inhibition by the classical ACE inhibitors [1,2]. After the elucidation of the crystal structure of testicular ACE (tACE), [19] a model of the active site of ACE2 was described which demonstrated the structural determinants underlying these differences in enzyme activity [17]. Critical residue substitutions were highlighted that gave rise to the elimination of the S2¢ pocket found in ACE such that ACE2 is able to remove only a single amino acid from the C-terminus of its substrates (whereas ACE is a peptidyl dipeptidase). Shortly after this, the structure of ACE2 was solved [20] which provided further insights into this enzyme in relation to its counterpart. However, it has 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 activesite 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, ...

show abstract

Arg1098 Is Critical for the Chloride Dependence of Human Angiotensin I-converting Enzyme C-domain Catalytic Activity

Cited by 57 publications

References 21 publications

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

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

Contact Info

Product

Resources

About