A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents

Bernstein, Kenneth E.; Welsh, Stephen L.; Inman, John K.

doi:10.1016/0006-291x(90)91766-l

Cited by 25 publications

(17 citation statements)

References 14 publications

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“…One prominent ␣-helix (helix 17, residues 511-531) connects the two subdomains and forms part of the floor of the canyon. The deeply recessed and shielded proteolytic active site of ACE2 is a common structural feature of proteases and exists to avoid hydrolysis of correctly folded and functional proteins (24,25) and is also consistent with the profiles of binding of tethered inhibitors to sACE (26,27).…”

Section: Resultssupporting

confidence: 68%

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

669

841

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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 ...

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Section: Resultssupporting

confidence: 68%

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

669

841

View full text Add to dashboard Cite

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“…ACE as having a deeply buried active site (Pantoliano et al, 1984;Bernstein et al, 1990). X-ray analysis confirmed the coordination of the zinc ion by His383, His387, and Glu411 (His959, His963, and Glu987 in somatic ACE).…”

Section: B Crystal Structure Of Angiotensinconverting Enzymementioning

confidence: 85%

“…X-ray and electron microscopic studies provide a detailed picture of the deep substrate-binding clefts suspected by earlier biochemical studies (Pantoliano et al, 1984;Bernstein et al, 1990). In this model of Fig.…”

Section: E Structure Of Angiotensin-converting Enzymementioning

confidence: 95%

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

et al. 2012

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“…Lisinopril was found to have an IC 50 of 1.9 nM, consistent with previous reports. 29 Following attachment of M-chelates to lisinopril, IC 50 values were found in the range 44 – 4,500 nM, with IC 50 values increasing (affinity decreasing) in the order M-NTA-lisinopril < M-GGH-lisinopril ~ M-EDTA-lisinopril < M-DOTA-lisinopril. These results confirm the expected inverse correlation between target affinity and steric bulk, as well as negative charge, of the attached M-chelate.…”

Section: Resultsmentioning

confidence: 99%

Targeted Catalytic Inactivation of Angiotensin Converting Enzyme by Lisinopril-Coupled Transition-Metal Chelates

2012

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A series of compounds that target reactive transition metal chelates to somatic Angiotensin Converting Enzyme (sACE-1) have been synthesized. Half maximal inhibitory concentrations (IC50) and rate constants for both inactivation and cleavage of full length sACE-1 have been determined and evaluated in terms of metal-chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine sidechain of lisinopril by EDC/NHS coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following pre-incubation with metal-chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal-chelate-lisinopril complexes revealed IC50 values ranging from 44 nM to 4,500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal-chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second order rate constants as high as 150,000 M−1min−1 (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primary from sidechain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal-chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.

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A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents

Cited by 25 publications

References 14 publications

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

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

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

Targeted Catalytic Inactivation of Angiotensin Converting Enzyme by Lisinopril-Coupled Transition-Metal Chelates

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