A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase
The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1‘ position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl...
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| Veröffentlicht in: | The Journal of biological chemistry 1991-02, Vol.266 (6), p.3827-3834 |
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| creator | Rosén, J Tomkinson, B Pettersson, G Zetterqvist, O |
| description | The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1‘ position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1‘ position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage. |
| doi_str_mv | 10.1016/S0021-9258(19)67868-3 |
| format | Article |
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During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1‘ position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(19)67868-3</identifier><identifier>PMID: 1995635</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Animals ; Autoradiography ; Biological and medical sciences ; Chromatography, DEAE-Cellulose ; Electrophoresis, Polyacrylamide Gel ; Endopeptidases - chemistry ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Humans ; Hydrolases ; Liver - enzymology ; Molecular Sequence Data ; Phosphoserine - chemistry ; Prolyl Oligopeptidases ; Protease Inhibitors - pharmacology ; Rats ; Serine Endopeptidases - chemistry ; Substrate Specificity</subject><ispartof>The Journal of biological chemistry, 1991-02, Vol.266 (6), p.3827-3834</ispartof><rights>1991 © 1991 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c464t-cd73fdfe2b190acc19e6860a7f3781d952b32473e444bf9e61da3fea2ec0c5ba3</citedby><cites>FETCH-LOGICAL-c464t-cd73fdfe2b190acc19e6860a7f3781d952b32473e444bf9e61da3fea2ec0c5ba3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19589220$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1995635$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rosén, J</creatorcontrib><creatorcontrib>Tomkinson, B</creatorcontrib><creatorcontrib>Pettersson, G</creatorcontrib><creatorcontrib>Zetterqvist, O</creatorcontrib><title>A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1‘ position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1‘ position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.</description><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Autoradiography</subject><subject>Biological and medical sciences</subject><subject>Chromatography, DEAE-Cellulose</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Endopeptidases - chemistry</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Hydrolases</subject><subject>Liver - enzymology</subject><subject>Molecular Sequence Data</subject><subject>Phosphoserine - chemistry</subject><subject>Prolyl Oligopeptidases</subject><subject>Protease Inhibitors - pharmacology</subject><subject>Rats</subject><subject>Serine Endopeptidases - chemistry</subject><subject>Substrate Specificity</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkVuL1TAUhYso45nRnzAQBG8w1dyaNk8yDN5gQEEF30Ka7k639KQ1SWc4v2n-pDnTg5cnAyEP61s7i72K4pTRV4wy9foLpZyVmlfNC6ZfqrpRTSnuFRtGG1GKin2_X2x-Iw-L4xh_0HykZkfFEdO6UqLaFLfnZFi21pMIAT0Q8N00w5ywsxHOyLwE7BE6coNpIAHiDC6RNBHrEl5j2hF7ZdHHRCxZbbCi8zDF_V2noidpAPKZPSfzFDHh5M8IRmLn2QbwadyR7PQJnR0P_jCNu_HfOI-KB70dIzw-vCfFt3dvv158KC8_vf94cX5ZOqlkKl1Xi77rgbdMU-sc06AaRW3di7phna54K7isBUgp2z6LrLOiB8vBUVe1VpwUz9a5OcTPBWIyW4wOxtF6mJZoGiqlYrLKYLWCLkwxBujNHHBrw84wavYlmbuSzL4Bw7S5K8mI7Ds9fLC0W-j-uNZWsv70oNuYN9IH6x3Gv7Cq0ZzTzD1ZuQGvhhsMYFqc3ABbw5UyyoiG1xl6s0KQV3aNEEx0CN5Blw0umW7C_8T9BfxYvqk</recordid><startdate>19910225</startdate><enddate>19910225</enddate><creator>Rosén, J</creator><creator>Tomkinson, B</creator><creator>Pettersson, G</creator><creator>Zetterqvist, O</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19910225</creationdate><title>A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase</title><author>Rosén, J ; Tomkinson, B ; Pettersson, G ; Zetterqvist, O</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c464t-cd73fdfe2b190acc19e6860a7f3781d952b32473e444bf9e61da3fea2ec0c5ba3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Autoradiography</topic><topic>Biological and medical sciences</topic><topic>Chromatography, DEAE-Cellulose</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Endopeptidases - chemistry</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Hydrolases</topic><topic>Liver - enzymology</topic><topic>Molecular Sequence Data</topic><topic>Phosphoserine - chemistry</topic><topic>Prolyl Oligopeptidases</topic><topic>Protease Inhibitors - pharmacology</topic><topic>Rats</topic><topic>Serine Endopeptidases - chemistry</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rosén, J</creatorcontrib><creatorcontrib>Tomkinson, B</creatorcontrib><creatorcontrib>Pettersson, G</creatorcontrib><creatorcontrib>Zetterqvist, O</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rosén, J</au><au>Tomkinson, B</au><au>Pettersson, G</au><au>Zetterqvist, O</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1991-02-25</date><risdate>1991</risdate><volume>266</volume><issue>6</issue><spage>3827</spage><epage>3834</epage><pages>3827-3834</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1‘ position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1‘ position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>1995635</pmid><doi>10.1016/S0021-9258(19)67868-3</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1991-02, Vol.266 (6), p.3827-3834 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Analytical, structural and metabolic biochemistry Animals Autoradiography Biological and medical sciences Chromatography, DEAE-Cellulose Electrophoresis, Polyacrylamide Gel Endopeptidases - chemistry Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Humans Hydrolases Liver - enzymology Molecular Sequence Data Phosphoserine - chemistry Prolyl Oligopeptidases Protease Inhibitors - pharmacology Rats Serine Endopeptidases - chemistry Substrate Specificity |
| title | A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase |
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