Pyridoxal-5′-phosphate-dependent catalytic antibodies
Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapt...
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description | Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten
N
α-(5′-phosphopyridoxyl)-
l-lysine, a stable analog of the covalent coenzyme–substrate adducts were screened by a competition ELISA for binding of the PLP–amino acid Schiff base adduct. The Schiff base with its C4′–Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic
d-amino acids and oxo acids (
k
cat′=0.42 min
−1 with
d-alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme–amino acid adduct. (2) The bond at Cα to be broken lies together with the CαN bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme–substrate adduct assumes a
cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10
4 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9
k
cat (Ab·PLP)′/
k
cat (PLP)′=5×10
3). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis. |
doi_str_mv | 10.1016/S0022-1759(02)00227-2 |
format | Article |
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N
α-(5′-phosphopyridoxyl)-
l-lysine, a stable analog of the covalent coenzyme–substrate adducts were screened by a competition ELISA for binding of the PLP–amino acid Schiff base adduct. The Schiff base with its C4′–Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic
d-amino acids and oxo acids (
k
cat′=0.42 min
−1 with
d-alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme–amino acid adduct. (2) The bond at Cα to be broken lies together with the CαN bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme–substrate adduct assumes a
cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10
4 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9
k
cat (Ab·PLP)′/
k
cat (PLP)′=5×10
3). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/S0022-1759(02)00227-2</identifier><identifier>PMID: 12379355</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Amino Acids - metabolism ; Analogy of catalytic antibody and corresponding enzyme ; Antibodies, Catalytic - chemistry ; Antibodies, Catalytic - metabolism ; Biological and medical sciences ; Catalysis ; Catalytic antibodies ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; Haptens - immunology ; Haptens - metabolism ; Humans ; Immunoglobulin Fab Fragments - chemistry ; Immunoglobulin Fab Fragments - metabolism ; Models, Molecular ; Molecular evolution ; Molecular immunology ; Protein Structure, Tertiary ; Pyridoxal Phosphate - metabolism ; Pyridoxal-5′-phosphate ; Techniques</subject><ispartof>Journal of immunological methods, 2002-11, Vol.269 (1), p.99-110</ispartof><rights>2002 Elsevier Science B.V.</rights><rights>2003 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c422t-7b5778c0254d1deed2bc74353d218508e202406387b26bf77be24c760aea8cf13</citedby><cites>FETCH-LOGICAL-c422t-7b5778c0254d1deed2bc74353d218508e202406387b26bf77be24c760aea8cf13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0022-1759(02)00227-2$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14574010$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12379355$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gramatikova, Svetlana</creatorcontrib><creatorcontrib>Mouratou, Barbara</creatorcontrib><creatorcontrib>Stetefeld, Jörg</creatorcontrib><creatorcontrib>Mehta, Perdeep K</creatorcontrib><creatorcontrib>Christen, Philipp</creatorcontrib><title>Pyridoxal-5′-phosphate-dependent catalytic antibodies</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten
N
α-(5′-phosphopyridoxyl)-
l-lysine, a stable analog of the covalent coenzyme–substrate adducts were screened by a competition ELISA for binding of the PLP–amino acid Schiff base adduct. The Schiff base with its C4′–Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic
d-amino acids and oxo acids (
k
cat′=0.42 min
−1 with
d-alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme–amino acid adduct. (2) The bond at Cα to be broken lies together with the CαN bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme–substrate adduct assumes a
cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10
4 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9
k
cat (Ab·PLP)′/
k
cat (PLP)′=5×10
3). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.</description><subject>Amino Acids - metabolism</subject><subject>Analogy of catalytic antibody and corresponding enzyme</subject><subject>Antibodies, Catalytic - chemistry</subject><subject>Antibodies, Catalytic - metabolism</subject><subject>Biological and medical sciences</subject><subject>Catalysis</subject><subject>Catalytic antibodies</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Haptens - immunology</subject><subject>Haptens - metabolism</subject><subject>Humans</subject><subject>Immunoglobulin Fab Fragments - chemistry</subject><subject>Immunoglobulin Fab Fragments - metabolism</subject><subject>Models, Molecular</subject><subject>Molecular evolution</subject><subject>Molecular immunology</subject><subject>Protein Structure, Tertiary</subject><subject>Pyridoxal Phosphate - metabolism</subject><subject>Pyridoxal-5′-phosphate</subject><subject>Techniques</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc9O3DAQh60KVBbaRyjiAmoPpuNJnMmeEFqVFgkJpLZny7EnwlU2CXYWsTeeiUfiScj-UffIaTTSN_MbfSPEFwXnClTx_TcAolSkp18Bv60akvhBTFRJKGkKek9M_iMH4jClfwCgoICP4kBhRtNM64mgu2UMvnuyjdSvzy-yv-9Sf28Hlp57bj23w4mzg22WQ3Anth1C1fnA6ZPYr22T-PO2Hom_Vz_-zH7Jm9uf17PLG-lyxEFSpYlKB6hzrzyzx8pRnunMoyo1lIyAORRZSRUWVU1UMeaOCrBsS1er7Eicbfb2sXtYcBrMPCTHTWNb7hbJECoqtX4fHOOUQpWNoN6ALnYpRa5NH8PcxqVRYFZqzVqtWXkzgGat1uA4d7wNWFRz9ruprcsRON0CNjnb1NG2LqQdl2vKxweM3MWG49HbY-BokgvcOvYhshuM78I7p7wB4euUyQ</recordid><startdate>20021101</startdate><enddate>20021101</enddate><creator>Gramatikova, Svetlana</creator><creator>Mouratou, Barbara</creator><creator>Stetefeld, Jörg</creator><creator>Mehta, Perdeep K</creator><creator>Christen, Philipp</creator><general>Elsevier B.V</general><general>Elsevier</general><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>7QO</scope><scope>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20021101</creationdate><title>Pyridoxal-5′-phosphate-dependent catalytic antibodies</title><author>Gramatikova, Svetlana ; Mouratou, Barbara ; Stetefeld, Jörg ; Mehta, Perdeep K ; Christen, Philipp</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c422t-7b5778c0254d1deed2bc74353d218508e202406387b26bf77be24c760aea8cf13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Amino Acids - metabolism</topic><topic>Analogy of catalytic antibody and corresponding enzyme</topic><topic>Antibodies, Catalytic - chemistry</topic><topic>Antibodies, Catalytic - metabolism</topic><topic>Biological and medical sciences</topic><topic>Catalysis</topic><topic>Catalytic antibodies</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Haptens - immunology</topic><topic>Haptens - metabolism</topic><topic>Humans</topic><topic>Immunoglobulin Fab Fragments - chemistry</topic><topic>Immunoglobulin Fab Fragments - metabolism</topic><topic>Models, Molecular</topic><topic>Molecular evolution</topic><topic>Molecular immunology</topic><topic>Protein Structure, Tertiary</topic><topic>Pyridoxal Phosphate - metabolism</topic><topic>Pyridoxal-5′-phosphate</topic><topic>Techniques</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gramatikova, Svetlana</creatorcontrib><creatorcontrib>Mouratou, Barbara</creatorcontrib><creatorcontrib>Stetefeld, Jörg</creatorcontrib><creatorcontrib>Mehta, Perdeep K</creatorcontrib><creatorcontrib>Christen, Philipp</creatorcontrib><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>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gramatikova, Svetlana</au><au>Mouratou, Barbara</au><au>Stetefeld, Jörg</au><au>Mehta, Perdeep K</au><au>Christen, Philipp</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Pyridoxal-5′-phosphate-dependent catalytic antibodies</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2002-11-01</date><risdate>2002</risdate><volume>269</volume><issue>1</issue><spage>99</spage><epage>110</epage><pages>99-110</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten
N
α-(5′-phosphopyridoxyl)-
l-lysine, a stable analog of the covalent coenzyme–substrate adducts were screened by a competition ELISA for binding of the PLP–amino acid Schiff base adduct. The Schiff base with its C4′–Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic
d-amino acids and oxo acids (
k
cat′=0.42 min
−1 with
d-alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme–amino acid adduct. (2) The bond at Cα to be broken lies together with the CαN bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme–substrate adduct assumes a
cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10
4 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9
k
cat (Ab·PLP)′/
k
cat (PLP)′=5×10
3). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>12379355</pmid><doi>10.1016/S0022-1759(02)00227-2</doi><tpages>12</tpages></addata></record> |
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subjects | Amino Acids - metabolism Analogy of catalytic antibody and corresponding enzyme Antibodies, Catalytic - chemistry Antibodies, Catalytic - metabolism Biological and medical sciences Catalysis Catalytic antibodies Fundamental and applied biological sciences. Psychology Fundamental immunology Haptens - immunology Haptens - metabolism Humans Immunoglobulin Fab Fragments - chemistry Immunoglobulin Fab Fragments - metabolism Models, Molecular Molecular evolution Molecular immunology Protein Structure, Tertiary Pyridoxal Phosphate - metabolism Pyridoxal-5′-phosphate Techniques |
title | Pyridoxal-5′-phosphate-dependent catalytic antibodies |
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