scholarly journals A tyrosine residue essential for catalytic activity in aminopeptidase A

1997 ◽  
Vol 327 (3) ◽  
pp. 883-889 ◽  
Author(s):  
Gilles VAZEUX ◽  
Xavier ITURRIOZ ◽  
Pierre CORVOL ◽  
Catherine LLORENS-CORTÈS
Keyword(s):  
Catalytic Activity ◽  
Transition State ◽  
Active Site ◽  
Consensus Sequence ◽  
Zinc Ion ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Tyrosine Residue ◽  
Aminopeptidase A

Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

2008 ◽  
Vol 389 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Branka Salopek-Sondi ◽  
Bojana Vukelić ◽  
Jasminka Špoljarić ◽  
Šumski Šimaga ◽  
Dušica Vujaklija ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transition State ◽  
Active Site ◽  
Functional Role ◽  
Dipeptidyl Peptidase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Tyrosine Residue ◽  
Transition State Stabilization ◽  
Endogenous Defense

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased k cat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.

scholarly journals Identification of an active site cysteine residue in human type I Ins(1,4,5)P3 5-phosphatase by chemical modification and site-directed mutagenesis

1996 ◽  
Vol 320 (1) ◽  
pp. 181-186 ◽  
Author(s):  
David COMMUNI ◽  
Christophe ERNEUX
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type I ◽  
Active Site Cysteine ◽  
Substrate Binding Domain ◽  
Active Site Cysteine Residue

Chemical modification using thiol-directed agents and site-directed mutagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human type I Ins(1,4,5)P3 5-phosphatase. Irreversible inhibition of enzymic activity is provoked by chemical modification of the enzyme by N-ethylmaleimide (NEM), 5,5´-dithio-2-nitrobenzoic acid, iodoacetate and to a much smaller extent by iodoacetamide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0.9 mol of NEM per mol of enzyme. A single [14C]NEM-modified peptide was isolated after α-chymotrypsin proteolysis of the radiolabelled enzyme and reverse-phase HPLC. Sequence analysis of the active site-labelled peptide (i.e. MNTRCPAWCD) demonstrated that Cys348 contained the radiolabel. Furthermore two mutant enzymes were obtained by site-directed mutagenesis of the cysteine residue to serine and alanine respectively. Both mutant enzymes had identical UV CD spectra. The two mutants (i.e. Cys348 → Ser and Cys348 → Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part of the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P3 5-phosphatase. This cysteine residue is part of a sequence 10 amino acids long that is well conserved among the primary structures of inositol and phosphatidylinositol polyphosphate 5-phosphatases.

scholarly journals Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue

1997 ◽  
Vol 326 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Gilles MILLAT ◽  
Roseline FROISSART ◽  
Irène MAIRE ◽  
Dominique BOZON
Keyword(s):  
Catalytic Activity ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Lysosomal Storage ◽  
Post Translational Modification ◽  
Cos Cells ◽  
Conserved Sequence ◽  
Glycosylation Sites

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271–278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.

Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine

2001 ◽  
Vol 360 (3) ◽  
pp. 657-665 ◽  
Author(s):  
Yong-Sun LEE ◽  
Young-Dong CHO
Keyword(s):  
Ornithine Decarboxylase ◽  
Active Site ◽  
Competitive Inhibition ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Three Dimensional Model ◽  
Nicotiana Glutinosa ◽  
Polyamine Biosynthesis ◽  
Conserved Sequence

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank® AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native Mr of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with Km values of 562μM and 1592μM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (Ki 1.15μM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys95) and cysteine (Cys96, Cys338 and Cys377) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys96, each mutation caused a substantial loss in enzyme activity. Most notably, Lys95 increased the Km for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the kcat for ODC and lysine decarboxylase (LDC) activity respectively. The Cys377 → Ala mutant possessed a kcat that was lowered by 23-fold, and the Km value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.

scholarly journals Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein fromEscherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

1998 ◽  
Vol 180 (18) ◽  
pp. 4799-4803 ◽  
Author(s):  
Frédérique Pompeo ◽  
Jean van Heijenoort ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acetyl Coa ◽  
Mutant Proteins ◽  
Acetyltransferase Activity ◽  
Phosphate Acetyltransferase ◽  
High Level

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis

1999 ◽  
Vol 341 (3) ◽  
pp. 805-812 ◽  
Author(s):  
Carl B. BARON ◽  
Dean R. TOLAN ◽  
Kyung H. CHOI ◽  
Ronald F. COBURN
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Bond Cleavage ◽  
Site Directed Mutagenesis ◽  
Binding Domains ◽  
Carbon Carbon Bond ◽  
Catalytically Active ◽  
Aldolase Activity ◽  
Aldolase A

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1,4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P3 binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P3 binding, increased EC50 values for Fru(1,6)P2-evoked release of bound Ins(1,4,5)P3 and increased Ki values for Ins(1,4,5)P3-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P3. Computer-simulated docking of Ins(1,4,5)P3 in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P3 to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P2 binding. Results indicate that Ins(1,4,5)P3 binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P3 and Fru(1,6)P2 for binding. Although Ins(1,4,5)P3 binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4,5)P3-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P3 in other proteins.

scholarly journals Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

2005 ◽  
Vol 386 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sandra MÜLLER ◽  
Manuela SCHÖTTLER ◽  
Sylvia SCHÖN ◽  
Christian PRANTE ◽  
Thomas BRINKMANN ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

scholarly journals Recombinant expression and isolation of human l-arginine:glycine amidinotransferase and identification of its active-site cysteine residue

1997 ◽  
Vol 322 (3) ◽  
pp. 771-776 ◽  
Author(s):  
Andreas HUMM ◽  
Erich FRITSCHE ◽  
Karlheinz MANN ◽  
Martin GÖHL ◽  
Robert HUBER
Keyword(s):  
Active Site ◽  
Recombinant Expression ◽  
Factor Xa ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cleavage Sites ◽  
Fusion Peptides ◽  
Kidney Mitochondria ◽  
Active Site Cysteine ◽  
Active Site Cysteine Residue

Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine: glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coliwith two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

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