scholarly journals ADAMDEC1 Is a Metzincin Metalloprotease with Dampened Proteolytic Activity

2013 ◽  
Vol 288 (29) ◽  
pp. 21367-21375 ◽  
Author(s):  
Jacob Lund ◽  
Ole H. Olsen ◽  
Esben S. Sørensen ◽  
Henning R. Stennicke ◽  
Helle H. Petersen ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Aspartic Acid ◽  
Proteolytic Activity ◽  
Active Site ◽  
Cleavage Site ◽  
Physiological Role ◽  
Aspartic Acid Residue ◽  
Glycosylation Sites ◽  
Proprotein Processing ◽  
Adam Family

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.

scholarly journals Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 β-lactamase II

1991 ◽  
Vol 276 (2) ◽  
pp. 401-404 ◽  
Author(s):  
H M Lim ◽  
R K Iyer ◽  
J J Pène
Keyword(s):  
Bacillus Cereus ◽  
Aspartic Acid ◽  
Carboxy Group ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Aspartic Acid Residue ◽  
Beta Lactamase Ii

An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an ‘in vivo’ study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

scholarly journals An aspartic acid residue at the active site of pepsin. The isolation and sequence of the heptapeptide

1969 ◽  
Vol 113 (2) ◽  
pp. 377-386 ◽  
Author(s):  
R. S. Bayliss ◽  
J. R. Knowles ◽  
Grith B. Wybrandt
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Irreversible Inhibitor ◽  
Aspartic Acid Residue ◽  
Phenylalanine Methyl Ester

Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser

scholarly journals An active-site peptide from pepsin C

1971 ◽  
Vol 123 (1) ◽  
pp. 75-82 ◽  
Author(s):  
J. Kay ◽  
A. P. Ryle
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Ph Values ◽  
Complete Inactivation ◽  
Active Site Sequence

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.

scholarly journals A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells

2003 ◽  
Vol 369 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Sanna PARTANEN ◽  
Stephan STORCH ◽  
Hans-Gerhard LÖFFLER ◽  
Andrej HASILIK ◽  
Jaana TYYNELÄ ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Active Site ◽  
Cathepsin D ◽  
Secretory Pathway ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Aspartic Acid Residue ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Lysosomal Protease ◽  
293 Cells

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786—2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Mariusz Olczak ◽  
Katarzyna Indyk ◽  
Teresa Olczak
Keyword(s):  
Proteolytic Activity ◽  
Active Site ◽  
Cleavage Site ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ph Optimum ◽  
Low Efficiency ◽  
Primary Cleavage ◽  
Secondary Cleavage

AbstractAzurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed inSf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.

scholarly journals Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

1991 ◽  
Vol 280 (2) ◽  
pp. 521-526 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Active Site ◽  
Citrate Synthase ◽  
Condensation Reaction ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Reverse Direction ◽  
Acetyl Coa ◽  
Aspartic Acid Residue

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp→Asn and Asp→Gly mutations. The Asp→Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

Isolation and characterization of a peptide derived from the active site of chicken pepsin

1980 ◽  
Vol 45 (7) ◽  
pp. 2131-2134 ◽  
Author(s):  
Helena Keilová ◽  
Vladimír Kostka ◽  
Miroslav Baudyš
Keyword(s):  
Amino Acid ◽  
Methyl Ester ◽  
Amino Acid Composition ◽  
Aspartic Acid ◽  
Acid Composition ◽  
Active Site ◽  
Its Sequence ◽  
Aspartic Acid Residue ◽  
Isolation And Characterization

A peptide was isolated from chicken pepsin which contains the aspartic acid residue reacting with diazoacetyl-D,L-norleucine methyl ester in the presence of Cu2+ -ions. The peptide is N-terminated with isoleucine and contains (besides isoleucine) valine, aspartic acid, two threonines, serine, and leucine. In concurrent experiments a peptide of the same composition was isolated from the thermolysin digest of chicken pepsin and its sequence determined as Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu. Since both peptides have entirely identical amino acid composition and other characteristics, the sequenced peptide corresponds to the peptide isolated from the active site of the enzyme.

scholarly journals Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic acid residue.

1994 ◽  
Vol 269 (18) ◽  
pp. 13680-13684
Author(s):  
A. Zlotnick ◽  
V.S. Reddy ◽  
R. Dasgupta ◽  
A. Schneemann ◽  
W.J. Ray ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Capsid Assembly ◽  
Aspartic Acid Residue

scholarly journals Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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