Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH
The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of NADPH with the two conformers differ by over 100-fold (K...
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| Veröffentlicht in: | The Journal of biological chemistry 1990-04, Vol.265 (10), p.5579-5584 |
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| container_title | The Journal of biological chemistry |
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| creator | APPLEMAN, J. R HOWELL, E. E KRAUT, J BLAKLEY, R. L |
| description | The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states,
Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of
NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion
of conformers are small (t1/2 for Ew---Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied
by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH
greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium
has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27
is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding
to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the
proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM
for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew---Et
(t1/2 = 221 s) and an increased rate for Et---Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR). |
| doi_str_mv | 10.1016/S0021-9258(19)39400-1 |
| format | Article |
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Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of
NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion
of conformers are small (t1/2 for Ew---Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied
by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH
greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium
has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27
is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding
to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the
proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM
for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew---Et
(t1/2 = 221 s) and an increased rate for Et---Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(19)39400-1</identifier><identifier>PMID: 2108144</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>Analytical, structural and metabolic biochemistry ; Aspartic Acid ; Binding Sites ; Biological and medical sciences ; dihydrofolate reductase ; Enzyme Activation ; Enzymes and enzyme inhibitors ; Escherichia coli - enzymology ; Fundamental and applied biological sciences. Psychology ; Kinetics ; Lactobacillus casei - enzymology ; Mutation ; NADP - metabolism ; NADPH ; Oxidoreductases ; Protein Conformation ; Streptococcus - enzymology ; Structure-Activity Relationship ; Tetrahydrofolate Dehydrogenase - metabolism</subject><ispartof>The Journal of biological chemistry, 1990-04, Vol.265 (10), p.5579-5584</ispartof><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c439t-29bef1647775753c3a705e4bf8813be00af37562917de3fc545a6be6fd79de83</citedby><cites>FETCH-LOGICAL-c439t-29bef1647775753c3a705e4bf8813be00af37562917de3fc545a6be6fd79de83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,782,786,27931,27932</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6942013$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2108144$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>APPLEMAN, J. R</creatorcontrib><creatorcontrib>HOWELL, E. E</creatorcontrib><creatorcontrib>KRAUT, J</creatorcontrib><creatorcontrib>BLAKLEY, R. L</creatorcontrib><title>Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states,
Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of
NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion
of conformers are small (t1/2 for Ew---Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied
by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH
greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium
has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27
is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding
to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the
proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM
for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew---Et
(t1/2 = 221 s) and an increased rate for Et---Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Aspartic Acid</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>dihydrofolate reductase</subject><subject>Enzyme Activation</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Escherichia coli - enzymology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Kinetics</subject><subject>Lactobacillus casei - enzymology</subject><subject>Mutation</subject><subject>NADP - metabolism</subject><subject>NADPH</subject><subject>Oxidoreductases</subject><subject>Protein Conformation</subject><subject>Streptococcus - enzymology</subject><subject>Structure-Activity Relationship</subject><subject>Tetrahydrofolate Dehydrogenase - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAQhi0EKkvhESpFAqFyCHhiO46PVSkUqQIEPXCzHGfcNUrsrZ1ttTwIz0uyG-0Vy5I1_r9_ZqSfkDOg74FC_eEnpRWUqhLNOah3THFKS3hCVkAbVjIBv56S1RF5Tl7k_JtOhys4ISfVRAHnK_L3R-yxiK4weWPSaEYsKjnXnV_vuhRd7Oe_hN3WjiZj4VIciqts15i8XXtT2Nj7wofpjphsDA-Yso9h39OO_gELE7pJXQoMf3YDTq7gYhomdi-3PnQ-3M2mrxcfv1-_JM-c6TO-Wt5Tcvvp6vbyurz59vnL5cVNaTlTY1mpFh3UXEoppGCWGUkF8tY1DbAWKTWOSVFXCmSHzFnBhalbrF0nVYcNOyVvD203Kd5vMY968Nli35uAcZu1VLWitYT_giBqDtDMHcUBtCnmnNDpTfKDSTsNVM-56X1ueg5Fg9L73PQ84GwZsG0H7I6uJahJf7PoJlvTu2SC9fmI1YpXFNiEvT5ga3-3fvQJdevjlNWgq1rMKwghFfsHjzWtXQ</recordid><startdate>19900405</startdate><enddate>19900405</enddate><creator>APPLEMAN, J. R</creator><creator>HOWELL, E. E</creator><creator>KRAUT, J</creator><creator>BLAKLEY, R. L</creator><general>American Society for Biochemistry and Molecular Biology</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>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19900405</creationdate><title>Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH</title><author>APPLEMAN, J. R ; HOWELL, E. E ; KRAUT, J ; BLAKLEY, R. L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c439t-29bef1647775753c3a705e4bf8813be00af37562917de3fc545a6be6fd79de83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Aspartic Acid</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>dihydrofolate reductase</topic><topic>Enzyme Activation</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli - enzymology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Kinetics</topic><topic>Lactobacillus casei - enzymology</topic><topic>Mutation</topic><topic>NADP - metabolism</topic><topic>NADPH</topic><topic>Oxidoreductases</topic><topic>Protein Conformation</topic><topic>Streptococcus - enzymology</topic><topic>Structure-Activity Relationship</topic><topic>Tetrahydrofolate Dehydrogenase - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>APPLEMAN, J. R</creatorcontrib><creatorcontrib>HOWELL, E. E</creatorcontrib><creatorcontrib>KRAUT, J</creatorcontrib><creatorcontrib>BLAKLEY, R. L</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</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>APPLEMAN, J. R</au><au>HOWELL, E. E</au><au>KRAUT, J</au><au>BLAKLEY, R. L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1990-04-05</date><risdate>1990</risdate><volume>265</volume><issue>10</issue><spage>5579</spage><epage>5584</epage><pages>5579-5584</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states,
Et and Ew, which differ in affinity for NADPH and in kinetic competence. Dissociation constants for the binary complex of
NADPH with the two conformers differ by over 100-fold (KDt = 0.17 microM, KDw = 22 microM). Rate constants governing the interconversion
of conformers are small (t1/2 for Ew---Et = 71 s), and since Ew is not catalytically competent, this conversion is accompanied
by an increase in catalytic velocity. The equilibrium proportion of Et in the absence of ligands is 63%, but binding of NADPH
greatly increases this proportion, and t1/2 for conversion of Ew.NADPH to Et.NADPH is 30 s. This conformational equilibrium
has also been examined in mutant enzyme in which aspartate 27 is replaced by asparagine (D27N E. coli DHFR). Although ASp27
is an active site residue, it does not interact directly with bound NADPH, and in the mutant the rate constant for NADPH binding
to Et is unchanged as are the dissociation constants for NADPH complexes with Et or Ew. However, for mutant apoenzyme, the
proportion of Et is decreased to 18% in the absence of ligands so that the overall KD for NADPH is increased (0.15 microM
for WT E. coli DHFR, 0.68 microM for D27N E. coli DHFR). The lower proportion of Et is due to a decreased rate for Ew---Et
(t1/2 = 221 s) and an increased rate for Et---Ew (t1/2 = 50 s versus 120 s for WT E. coli DHFR).</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>2108144</pmid><doi>10.1016/S0021-9258(19)39400-1</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1990-04, Vol.265 (10), p.5579-5584 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_79690671 |
| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Analytical, structural and metabolic biochemistry Aspartic Acid Binding Sites Biological and medical sciences dihydrofolate reductase Enzyme Activation Enzymes and enzyme inhibitors Escherichia coli - enzymology Fundamental and applied biological sciences. Psychology Kinetics Lactobacillus casei - enzymology Mutation NADP - metabolism NADPH Oxidoreductases Protein Conformation Streptococcus - enzymology Structure-Activity Relationship Tetrahydrofolate Dehydrogenase - metabolism |
| title | Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH |
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