Angiotensin I-converting Enzyme Transition State Stabilization by His1089

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

doi:10.1074/jbc.m009009200

Cited by 34 publications

(28 citation statements)

References 30 publications

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“…The much larger impact on k cat compared with K m supports a catalytic role for this histidine rather than an important binding function (44). His 505 appears to be too far from the zinc-bound carboxylate (6.3 Å) to be directly involved in H-bonding stabilization of the carbonyl tetrahedral intermediate, as was expected from sequence homology (44). Protonation and stabilization of the sp 3 hybridized nitrogen of the peptide intermediate as depicted in Fig.…”

Section: Resultsmentioning

confidence: 78%

“…A protonated histidine is supported by the pH-rate profile that has a maximum at 6.5 (8). Additional evidence for the role for His 505 comes from site-directed mutagenesis in sACE, where mutation of the analogous His 1089 to Ala results in a 100-fold drop in k cat and 4.5-fold drop in K m (44). Similarly, the His 1089 3 Leu mutation results in a 671-fold drop in k cat and a 6.2-fold drop in K m .…”

Section: Resultsmentioning

confidence: 90%

See 1 more Smart Citation

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

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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: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 90%

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

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“…Gln stabilizes the transition-state oxyanion in papain (22). His has a five-membered ring similar to the indole ring of Trp, with a nitrogen atom in a similar position, and has been shown to stabilize the transition-state oxyanion in metalloproteases (23). Tyr, like Trp, has a ring structure, which may participate in hydrophobic and aromatic interactions, and a hydroxyl group that potentially stabilizes the oxyanion in zinc metallopeptidases (24) by hydrogen bonding.…”

Section: Tyr Partially Compensates For Trp In Transition-state Stabilmentioning

confidence: 99%

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Iismaa

Holman

Wouters

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N 1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant Km (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant kcat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

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“…1. The gene constructs were made by strategies reported previously (11,12), chemically synthesized and cloned into the shuttle expression vector pcDNA3 (Invitrogen). The C-domain gene construct encodes amino acids Ϫ29 to Ϫ1 (signal peptide), 1 to 4, and 611 to 1201 of human somatic ACE (14) with an 8-residue FLAG epitope recognized by a commercially available antibody (M2, Sigma) at the C terminus.…”

Section: Construction Of Human Ace C-and N-domain Gene Constructs-mentioning

confidence: 99%

“…ACE was not further purified to remove the contaminating bovine serum albumin since the stability of the pure (Ͼ95%) Cdomain at 4°C was markedly lower than that of partially purified ACE containing bovine serum albumin. The concentration of enzyme in each crude or purified C-or N-domain or mutant preparation was quantified by Western blot analysis using a polyclonal antiserum generated against pure human kidney ACE (11).…”

Section: Construction Of Human Ace C-and N-domain Gene Constructs-mentioning

confidence: 99%

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

Liu¹,

Fernandez²,

Wouters³

et al. 2001

Journal of Biological Chemistry

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Angiotensin (Ang) I-converting enzyme (ACE) is a Zn2؉ metalloprotease with two homologous catalytic domains. Both the N-and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl ؊ , but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl ؊ dependence. Angiotensin I (Ang I) 1 -converting enzyme (ACE, EC 3.4.15.1) belongs to the gluzincin family (clan MA) of metalloproteases and is a peptidyl dipeptidase with broad substrate specificity (1). Peptide hydrolysis is activated by monovalent anions such as Cl Ϫ ; this feature is unique among metalloproteases (2). The somatic form of human ACE has two homologous catalytic domains. These N-and C-domains most likely are the result of an ancient gene duplication event that occurred during vertebrate evolution (3, 4). Invertebrate ACE has a single, Cl Ϫ -sensitive catalytic domain (5). The physiological substrates of ACE include Ang I, bradykinin, substance P, and AcSDKP (2); however, there are significant differences between the catalytic domains in catalytic efficiencies for the hydrolysis of these substrates (6, 7).Cl Ϫ dependence of hydrolysis is substrate-specific. For example, the degree of enzyme activation by Cl Ϫ and its apparent dissociation constant (K d,app ) associated with this activation is high for Ang I and low for bradykinin (8, 9). To define the molecular determinants in the substrate structure responsible for these differences, Riordan and colleagues (8, 9) studied the hydrolysis of a collection of tripeptide substrates with varying C-terminal dipeptide structures. They observed that a P 1 Ј-or P 2 Ј-(Arg/Lys) 2 in the tripeptide substrate was necessary and sufficient for high affinity Cl Ϫ binding. Bradykinin, like these substrates, but unlike Ang I, contains a basic residue in its C-terminal dipeptide. Also, hydrolytic activity of ACE for tripeptide substrates with a P 1 Ј-or P 2 Ј-(Arg/Lys) is less affected by Cl Ϫ than for those that lack a basic P 1 Ј or P 2 Ј residue. Because chemical modification of ACE that leads to methylation of Lys residues has selective effects on hydrolytic activity for furanacryloyl-FGG-COO Ϫ (inactivated by Ͼ99%) and furanacryloyl-FFR-COO Ϫ (activity reduced by ϳ50%) it was proposed that a critical Lys is part of the Cl Ϫ -binding site of ACE (10). The study by Shapiro and Riordan (10) had provided only circumstantial evidence that a critical Lys was involved in Cl Ϫ binding, and in principle it is best to consider both types of basic residues as possibilities. To identify the Cl Ϫ -binding residue(s) in ACE we made single substitutions with Gln of all conserved arginines or lysines in the human ACE C-domain. Here we show that the primary site of Cl Ϫ binding in the C-domain is Arg 1098 irrespective of whether the substrate is the decapeptide Ang I or a short peptide with or without a basic residue a...

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Angiotensin I-converting Enzyme Transition State Stabilization by His1089

Cited by 34 publications

References 30 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

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

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

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