Reactive Serine Residue Research Articles

Crystal structure of the essential Mycobacterium tuberculosis phosphopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein.

Phosphopantetheinyl transferases (PPTases) are key enzymes in the assembly-line production of complex molecules such as fatty acids, polyketides and polypeptides, where they activate acyl or peptidyl carrier proteins, transferring a 4'-phosphopantetheinyl moiety from coenzyme A (CoA) to a reactive serine residue on the carrier protein. The human pathogen Mycobacterium tuberculosis encodes two PPTases, both essential and therefore attractive drug targets. We report the structure of the type-II PPTase PptT, obtained from crystals of a fusion protein with maltose binding protein. The structure, at 1.75Å resolution (R=0.156, Rfree=0.191), reveals an α/β fold broadly similar to other type-II PPTases, but with differences in peripheral structural elements. A bound CoA is clearly defined with its pantetheinyl arm tucked into a hydrophobic pocket. Interactions involving the CoA diphosphate, bound Mg(2+) and three active site acidic side chains suggest a plausible pathway for proton transfer during catalysis.

Purification, properties and developmental regubdion of a Ca 2+-independent serine—cysteine protease from Allomyces arbuscula

Reproductive differentiation in Allomyces takes place against the background of substrate limitation, a sharp increase in intracellular probeolysine and the induction of at least one specific protease. The aim of this report is to describe the purification, properties and developmental regulation of this enzyme. The enzyme has been partially pied by a combination of ion exchange chromatography, ultrogel filtration and amity chromatography. The purified enzyme in SDS-PAGE appeared as a doublet of M r 40–43 kDa. Two bands corresponding to a relative molecular mass of 40–43 kDa were also apparent in activity gels. The protein has an apparent molecular mass in the region of 43 kDa as estimated by gel gyration. Th enzyme therefore, seems to be a monomer of 43 kDa. Tie second band in SDS-PAGE and activity gels Is probably the proteoiyzed form of the enzyme. The protease recognized alanine and to a lesser extant pbmylalunine in the P 1 position when assayed with a range of synthetic peptides. The wave site of the enzyme detains a reactive serine residue, as own by its labibition with PMSF and soya bean trypsin inhibitor. There is probably a reactive cystaine residue as well since the enzyme activity is strongly inhibited by HgC1 2, a thiol group binding reagent. The enzyme is present in zoospores but disappears progressively during germination and hypbal growth. It reappears when actively growing cultures are transferred to dilate sale solution. In conclusion, we have pied a serine-cysteine protease of M r 43 kDa. This enzyme has a very restricted substrate specificity and appears to be developmentally regulated.

Prolyl Endopeptidase from Flavobacterium meningosepticum: Cloning and Sequencing of the Enzyme Gene1

The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].

Protease II from Escherichia coli: Sequencing and Expression of the Enzyme Gene and Characterization of the Expressed Enzyme1

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.

The reactive serine residue of epidermolytic toxin A

Comparison of amino acid sequence data suggested that there may be a functional relationship between the staphylococcal epidermolytic toxins and V8 proteinase. The hypothesis was tested by treating epidermolytic toxin with di-isopropyl phosphorofluoridate, which bound specifically at serine-195, the homologue of the active-site serine residue of V8 proteinase.

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin.

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.

The structure and function of carboxypeptidase CN.

Carboxypeptidase CN from Citrus natsudaidai HAYATA is an acidic protease (M, 93000). The enzyme was inactivated by the incorporation of 1.0 mol of 32P-labeled diisopropylphosphofluoridate, 1.3 mol of 203HgCl2, or 2.2 mol of 110mAgNO3 per mol of enzyme at pH 5.5. Four or five radioactive peptides were isolated from a partial acid hydrolysate of the 32P-labeled enzyme. These peptides provided evidence indicating the amino acid sequence Glx-Gly-Asx-Ser-Gly-Gly-Glu-Leu-Val around the reactive serine residue. The enzyme reacted with 0.4 mol of 203Hg-labeled p-chloromercuribenzoate without appreciable loss of enzymatic activity. On the other hand, 1 mol of 203Hg-labeled p-chloromercuribenzoate was incorporated into the enzyme which had been denatured with 6 M guanidine hydrochloride and 8 M urea at pH 7.0 in the absence of dithiothreitol. Thus, the enzyme has one sulfhydryl group which possesses only poor reactivity. Photooxidation using methylene blue as a sensitizer caused a loss of enzymatic activity and specific destruction of approximately 1 mol of histidine residue per mol of enzyme. The photooxidized, p-bromophenacyl bromide-treated and HgCl2-treated enzyme failed to react with 32P-labeled diisopropylphosphofluoridate. From these findings, we inferred that one serine, one histidine, and the carboxyl groups are essential for catalytic activity.

Amino acid sequences of the amino- and carboxyl-terminal and reactive serine regions of carboxypeptidase CUa.

The amino acid sequences of the amino-and carboxyl-terminal regions of carboxypeptidase CUa were determined to be Ala-Val-Glu-Leu-His-Phe-Ile-His-Asn-and-Arg-His-Met-Glu-Pro-(Ala, Asp, Lys)-Gly-Thr-Ser, respectively. The enzyme was inactivated with the incorporation of 1 mol of 3H-labeled diisopropylfluorophosphate (DFP) per mol of enzyme, and this reagent was found to react with a serine residue in the active site of the enzyme. The primary structure around this reactive serine residue was determined to be Asp-Val-Ala-Gly-Tyr-Asp-Ser-Glu-Trp-Ile-Gln-Leu-Arg-Val-Pro-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Glu-Leu-Asp-Lys-Glu-Gly-Met-Ala-Pro-Asn-Gly-Ile-Val-Ser-Asp-Ala-Leu-Phe-Thr-Ser-Arg (Ser, reactive serine) by sequence analysis of two radioactive peptides released from [3H] DFP-treated carboxypeptidase CUa on tryptic digestion and cyanogen bromide cleavage.

The active site of carboxypeptidase CU. I. Evidence for serine in the active sites of carboxypeptidases CUa and CUb.

Carboxypeptidases CUa and CUb were both inactivated with incorporation of 1 mol 32P-labeled diisopropyl fluorophosphate per mol enzyme. The amino acid residue that reacted with this reagent was a serine residue in the active site of each enzyme. The amino acid sequence around this reactive serine residue was determined to be Glu-Gly-Asp-Ser-Gly-Gly-Glu-Leu for both enzymes by sequence analysis of three radioactive peptides isolated from partial acid hydrolysates of the 32P-labeled enzymes.

The amino acid sequences around the reactive serine and histidine residues of the chymotrypsin-like protease from the hornet, Vespa orientalis

The endopeptidase II from the larva of the hornet Vespa orientalis was inactivated by radioactively labeled active-site-directed inhibitors and afterwards digested with trypsin and chymotrypsin. The labeled peptides were isolated and sequenced. The peptide around the reactive serine residue contains 39 amino acids, and 30 amino acids constitute the peptide around the essential histidine residue. These peptides show sequence homology to the corresponding ones of the trypsin-related endopeptidases.

Human plasma kallikrein. A rapid purification method with high yield.

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).

Reactive serine in human adenovirus hexon polypeptide

Affinity labeling with diisopropylfluorophosphate (DFP) is used to study the enzymes associated with human adenovirus type 2 infection. A DFP-labeled 120K polypeptide is constantly found at a late stage of the virus cycle. Biochemical and immunological data indicate that this 120K polypeptide corresponds to the hexon subunit. No detectable enzymatic activity is found associated with native or urea-denatured adenovirus 2 hexon capsomer, and no DFP-labeled components appeared to be incorporated into virus or assembly intermediate particles. DFP reacts with urea-denatured hexon in vitro with an apparent dissociation constant of 5 × 10 -9 M. The occurrence of at least one transient reactive serine residue on the hexon polypeptide subunit and its possible implication in folding and/or assembly of hexon subunits into a hexon trimer is discussed.

The active site of an inducible arylacylamidase from Pseudomonas acidovorans.

Though very effectively ibhibited by both SH-group directed reagents and inhibitors typical for serine hydrolases, the inducible arylacylamidase from Pseudomonas acidovarans is a serine enzyme rather than a sulfhydryl enzyme. The amino acid sequence around the reactive serine residue is Gly-Ser-Ile. The sequence of a larger peptide from the active site is described.

Serine at the Active Center of Yeast Carboxypeptidase

Abstract Carboxypeptidase Y from bakers' yeast is a diisopropyl phosphorofluoridate (DFP)-sensitive enzyme. The inactivation by [32P]DFP is accompanied by the formation of 1 mole of labeled serine per mole of enzyme. A 15-residue 32P-labeled (*) peptide has been isolated from a peptic digest and shown to have the sequence His-Ile-Ala-Gly-Glu-Ser*-Tyr-Ala-His-Gly-Tyr-Ile-Pro-Val-Phe. The reaction of the serine residue with DFP is blocked by the presence of p-hydroxymercuribenzoate, which also inactivates the enzyme. The single —SH group of the protein may be near the active center of the enzyme, but the —SH group is not available to iodoacetate or iodoacetamide in the absence of denaturants. The sequence around the reactive serine residue and the properties of the —SH group contribute to an active site which is quite different from that of DFP-sensitive proteinases such as trypsin, chymotrypsin, or subtilisin.

The reactive serine residue in phosphoglucomutase of Micrococcus lysodeikiticus.

1. Phosphoglucomutase of Micrococcus lysodeikticus was labelled at the active site by exchange with (32)P-labelled substrates of high specific radioactivity. 2. Partial acid hydrolysis gave rise to radioactive peptides; serine phosphate was identified as one of the derivatives. 3. Comparison of the other (32)P-labelled peptides with the peptides obtained from the (32)P-labelled rabbit muscle phosphoglucomutase indicates that the sequence around the reactive serine residue is identical in both enzymes.

Mechanism of Action of α-1-Antitrypsin

Comparison of the biochemical characteristics of enzymes inhibited by α-1-antitrypsin shows that thrombin, elastase, trypsin, chymotrypsin, and plasmin are serine proteases with serine, histidine, and aspartic acid residues at their active center and cleave proteins at lysine and arginine or at aromatic or leucine residues. The present study was designed to test the hypothesis that α-1-antitrypsin has two inhibitor sites, one containing a positively charged residue and the other containing an aromatic or leucine residue, and that both sites have a binding site for the active site of serine proteases. Experiments designed to test this hypothesis showed the following: 1. Trypsin and chymotrypsin compete for inhibitory sites on purified α-1-antitrypsin, suggesting that the inhibitor has the same or overlapping inhibitory sites for these two enzymes. 2. Trypsin inactivated by diisopropylphosphorofluoridate fails to occupy inhibiting sites on α-1-antitrypsin. 3. α-1-Antitrypsin inhibits subtilisin, a proteolytic enzyme that contains serine, histidine, and aspartic acid residues at its active site in common with mammalian serine proteases. 4. α-1-Antitrypsin fails to inactivate acetylcholinesterase, a nonproteolytic enzyme whose active site contains a reactive serine residue, suggesting that this residue alone is not sufficient for inhibition by α-1-antitrypsin. 5. Treatment of α-1-antitrypsin with phenylglyoxal hydrate blocks the action of α-1-antitrypsin on trypsin but not on chymotrypsin. The change in activity is presumptive evidence that arginine residues were modified by the treatment. Antitryptic activity can be regenerated with removal of the blocking groups. These data indicate that trypsin and chymotrypsin are inhibited at two different sites on α-1-antitrypsin and suggest that the trypsin inhibitory site contains a positively charged amino acid. While these experiments have not proved this hypothesis, they are consistent with it.

Amino acid sequence in the region of the reactive serine residue of eel acetylcholinesterase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleAmino acid sequence in the region of the reactive serine residue of eel acetylcholinesteraseNorwood K. Schaffer, Harry O. Michel, and Alec F. BridgesCite this: Biochemistry 1973, 12, 15, 2946–2950Publication Date (Print):July 1, 1973Publication History Published online1 May 2002Published inissue 1 July 1973https://doi.org/10.1021/bi00739a027RIGHTS & PERMISSIONSArticle Views52Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (570 KB) Get e-Alerts Get e-Alerts

Amino acid sequence in the vicinity of the reactive serine residue in shrimp trypsin

The amino acid sequence in the vicinity of the DFP-sensitive serine residue in shrimp trypsin has been determined. Homologies with the serine active centers of proteases from the pancreas of several other species were noted. The results indicate a strong tendency to conserve certain portions of the sequence around the reactive serine residue despite the fact that mammals and crustaceans diverged at least 500 million years ago.

Radiochemical determination of a unique sequence around the reactive serine residue of a di-isopropyl phosphorofluoridate-sensitive plant carboxypeptidase and a yeast peptidase.

Phaseolain, a carboxypeptidase from French-bean leaves, and a partially purified peptidase from baker's yeast are inhibited by reaction with di-isopropyl phosphorofluoridate. Radioactive di-isopropyl [(32)P]phosphorofluoridate was used to show that the site of reaction is a unique serine residue and that the sequence of amino acids adjacent to the reactive serine is Glu-Ser-Tyr. This sequence is different from those of other ;serine' enzymes previously reported and, for phaseolain, represents an unequivocal example of a ;serine' carboxypeptidase.