scholarly journals Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity

1996 ◽  
Vol 318 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Tracy A. WILLIAMS ◽  
Annie MICHAUD ◽  
Xavier HOUARD ◽  
Marie-Thérèse CHAUVET ◽  
Florent SOUBRIER ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Pichia Pastoris ◽  
Ace Inhibitors ◽  
Active Sites ◽  
Chloride Concentration ◽  
Enzymic Activity ◽  
Expression Level ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Angiotensin I Converting Enzyme

Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant. Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCEΔCHO). This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin I and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE. However, AnCEΔCHO was less stable, displaying a half-life of 4.94 h at 37 °C, compared with AnCE which retained full activity under the same conditions. Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis. A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.

Catalytic properties of recombinant dipeptidyl carboxypeptidase from Escherichia coli: a comparative study with angiotensin I-converting enzyme

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Carlos Eduardo L. Cunha ◽  
Helena de Fátima Magliarelli ◽  
Thaysa Paschoalin ◽  
Aloysius T. Nchinda ◽  
Jackson C. Lima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ace Inhibitors ◽  
Catalytic Properties ◽  
Resonance Energy ◽  
Human Testis ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Acceptor Pair ◽  
Angiotensin I Converting Enzyme ◽  
Dipeptidyl Carboxypeptidase

Abstract Dipeptidyl carboxypeptidase from Escherichia coli (EcDcp) is a zinc metallopeptidase with catalytic properties closely resembling those of angiotensin I-converting enzyme (ACE). However, EcDcp and ACE are classified in different enzyme families (M3 and M2, respectively) due to differences in their primary sequences. We cloned and expressed EcDcp and studied in detail the enzyme's S3 to S1′ substrate specificity using positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides. These peptides contain ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as donor/acceptor pair. In addition, using FRET substrates developed for ACE [Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH] as well as natural ACE substrates (angiotensin I, bradykinin, and Ac-SDKP-OH), we show that EcDcp has catalytic properties very similar to human testis ACE. EcDcp inhibition studies were performed with the ACE inhibitors captopril (K i=3 nm) and lisinopril (K i=4.4 μm) and with two C-domain-selective ACE inhibitors, 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW; K i=22.0 μm) and lisinopril-Trp (K i=0.8 nm). Molecular modeling was used to provide the basis for the differences found in the inhibitors potency. The phylogenetic relationship of EcDcp and related enzymes belonging to the M3 and M2 families was also investigated and the results corroborate the distinct origins of EcDcp and ACE.

scholarly journals Effect of temperature and chloride on steady-state inhibition of angiotensin I-converting enzyme by enalaprilat and ramiprilat

1990 ◽  
Vol 272 (2) ◽  
pp. 415-419 ◽  
Author(s):  
A Skoglof ◽  
P O Göthe ◽  
J Deinum
Keyword(s):  
Steady State ◽  
Amino Acid ◽  
Protein Concentration ◽  
Chloride Concentration ◽  
Effect Of Temperature ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Inhibitor Binding ◽  
Angiotensin I Converting Enzyme ◽  
Kinetics Of

The kinetics of the steady-state inhibition of angiotension I-converting enzyme (EC 3.4.15.1) at 25 degrees C and 37 degrees C with enalaprilat and ramiprilat can be simulated, assuming only one inhibitor-binding site, consistent with a 1:1 stoichiometry if the protein concentration was determined by amino acid analysis. In this temperature range the apparent inhibition constants for ramiprilat and enalaprilat were roughly doubled by a decrease in the chloride concentration from 0.300 M to 0.120 M.

scholarly journals Association of angiotensin I-converting enzyme gene polymorphism with susceptibility to antiproteinuric effect of angiotensin I-converting enzyme inhibitors in patients with proteinuria.

1995 ◽  
Vol 6 (6) ◽  
pp. 1676-1678
Author(s):  
T Moriyama ◽  
H Kitamura ◽  
S Ochi ◽  
M Izumi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Ace Inhibitors ◽  
Urinary Protein ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Ace Gene ◽  
Protein Excretion ◽  
Angiotensin I Converting Enzyme ◽  
Antiproteinuric Effect ◽  
The Difference ◽  
Effective Group

The antiproteinuric effect of angiotensin I-converting enzyme (ACE) inhibitors in patients with renal diseases of various origins has been well recognized. However, individual responses regarding the degree of decrease in urinary protein excretion appear to vary considerably. The mechanism underlying this variable response to ACE inhibitors has not been clarified yet. A possible role of ACE gene insertion/deletion (I/D) polymorphism in the responsiveness to antiproteinuric effect of ACE inhibitors is examined. Thirty-six patients with proteinuria (23 men and 13 woman; mean age, 47 +/- 13 yr) were studied. These patients were classified into two groups on the basis of the percent decrease in their urinary protein excretion: the effective group, those with a decrease in proteinuria (18 patients, -64 +/- 19%) and the noneffective group (18 patients, +13 +/- 40%). A 287-base pair (bp) I/D polymorphism in the ACE gene was examined by polymerase chain reaction. The allelic frequencies of the ACE gene were I/D = 0.53/0.47 in the effective group and I/D = 0.81/0.19 in the noneffective group. The difference in the allelic frequencies between the two groups was significant (chi 2 = 6.25, P = 0.0114 < 0.05). Furthermore, the difference in the responsiveness of proteinuria to ACE inhibition between genotype II versus genotype ID + DD was statistically significant (chi 2 = 4.05, P = 0.0442 < 0.05). There was no significant difference between the two groups with regard to initial urinary protein level, blood pressure, renal function, and daily sodium intake. The genetic susceptibility to the antihypertensive effect of ACE inhibitors was also studied, but no significant relation was observed. This study suggests the association of ACE gene I/D polymorphism with the antiproteinuric efficacy of ACE inhibitors in patients with proteinuria.

SPECIFICITY EVALUATION OF THE TWO ACTIVE SITES OF ANGIOTENSIN I CONVERTING ENZYME USING COMBINATORIAL FLUORESCENT-QUENCHED PEPTIDE LIBRARIES

2004 ◽  
Vol 22 (Suppl. 1) ◽  
pp. S176
Author(s):  
Patricia Alessandra Bersanetti ◽  
M. C.C. Andrade ◽  
M. A. Juliano ◽  
D. E. Casarini ◽  
E. D. Sturrock ◽  
...  
Keyword(s):  
Active Sites ◽  
Peptide Libraries ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Angiotensin I Converting Enzyme
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