Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex
Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The...
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| Veröffentlicht in: | The Journal of biological chemistry 1992-06, Vol.267 (18), p.13028-13038 |
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| creator | BRZOVIC, P. S SAWA, Y HYDE, C. C MILES, E. W DUNN, M. F |
| description | Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations
in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit
active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe
to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for
Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady
state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction.
However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are
observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for
each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution
into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., BrzoviÄ, P., Drewe, W.F.,
Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are
part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced
inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed"
structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of
indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence
of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open
to the closed form. |
| doi_str_mv | 10.1016/S0021-9258(18)42377-0 |
| format | Article |
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in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit
active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe
to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for
Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady
state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction.
However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are
observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for
each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution
into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., BrzoviÄ, P., Drewe, W.F.,
Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are
part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced
inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed"
structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of
indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence
of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open
to the closed form.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)42377-0</identifier><identifier>PMID: 1618800</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>Allosteric Regulation ; alpha subunit ; Analytical, structural and metabolic biochemistry ; Base Sequence ; beta subunit ; Binding Sites ; biological activity ; Biological and medical sciences ; DNA ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Indoles - metabolism ; Kinetics ; Lyases ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Protein Conformation ; Salmonella typhimurium ; Salmonella typhimurium - enzymology ; site-directed mutagenesis ; Spectrophotometry, Ultraviolet ; tryptophan synthase ; Tryptophan Synthase - chemistry ; Tryptophan Synthase - genetics ; Tryptophan Synthase - metabolism ; U.V. spectroscopy</subject><ispartof>The Journal of biological chemistry, 1992-06, Vol.267 (18), p.13028-13038</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c440t-4ccfa87cd6fa7c3aba95bf0ec6181557baa2de8e40c87dfff79d5ff2a04ea3f43</citedby><cites>FETCH-LOGICAL-c440t-4ccfa87cd6fa7c3aba95bf0ec6181557baa2de8e40c87dfff79d5ff2a04ea3f43</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=5451116$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1618800$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>BRZOVIC, P. S</creatorcontrib><creatorcontrib>SAWA, Y</creatorcontrib><creatorcontrib>HYDE, C. C</creatorcontrib><creatorcontrib>MILES, E. W</creatorcontrib><creatorcontrib>DUNN, M. F</creatorcontrib><title>Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations
in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit
active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe
to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for
Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady
state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction.
However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are
observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for
each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution
into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., BrzoviÄ, P., Drewe, W.F.,
Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are
part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced
inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed"
structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of
indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence
of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open
to the closed form.</description><subject>Allosteric Regulation</subject><subject>alpha subunit</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>beta subunit</subject><subject>Binding Sites</subject><subject>biological activity</subject><subject>Biological and medical sciences</subject><subject>DNA</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Indoles - metabolism</subject><subject>Kinetics</subject><subject>Lyases</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Protein Conformation</subject><subject>Salmonella typhimurium</subject><subject>Salmonella typhimurium - enzymology</subject><subject>site-directed mutagenesis</subject><subject>Spectrophotometry, Ultraviolet</subject><subject>tryptophan synthase</subject><subject>Tryptophan Synthase - chemistry</subject><subject>Tryptophan Synthase - genetics</subject><subject>Tryptophan Synthase - metabolism</subject><subject>U.V. spectroscopy</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkcFu1DAQhi0EKkvhESr5gBA9BOwkTpwjqkpBqsQBkLhZE2fcGCV2sB1geSCeE2d3tfhiyfP984_nJ-SKszec8ebtZ8ZKXnSlkK-5vK7Lqm0L9ojsOJNVUQn-7THZnZGn5FmM31k-dccvyAVvuJSM7cjf2592QKeRphESndcEyXoXqXUU6OT9QgM-5BfqTUaQwrSMUMS1X51NmRptn--tkgK4aDc1NcHPFLJmQUeTz5305CMOVHtnfJgPHpvFUbdfks9dHY17l8eISHuL7s9-xiyYlwl_PydPDEwRX5zuS_L1_e2Xmw_F_ae7jzfv7gtd1ywVtdYGZKuHxkCrK-ihE71hqPN3uRBtD1AOKLFmWraDMabtBmFMCaxGqExdXZJXx75L8D9WjEnNNmqcJnDo16h4U8mOC5ZBcQR18DEGNGoJdoawV5ypLR91yEdty1dcqkM-atNdnQzWfsbhv-oYSK6_PNUhaphM3qm28YyJWnCehzhjo30Yf9mAqrdejzirsmk3P16xUlb_ANZkqaI</recordid><startdate>19920625</startdate><enddate>19920625</enddate><creator>BRZOVIC, P. S</creator><creator>SAWA, Y</creator><creator>HYDE, C. C</creator><creator>MILES, E. W</creator><creator>DUNN, M. F</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></search><sort><creationdate>19920625</creationdate><title>Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex</title><author>BRZOVIC, P. S ; SAWA, Y ; HYDE, C. C ; MILES, E. W ; DUNN, M. F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c440t-4ccfa87cd6fa7c3aba95bf0ec6181557baa2de8e40c87dfff79d5ff2a04ea3f43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Allosteric Regulation</topic><topic>alpha subunit</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Base Sequence</topic><topic>beta subunit</topic><topic>Binding Sites</topic><topic>biological activity</topic><topic>Biological and medical sciences</topic><topic>DNA</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Indoles - metabolism</topic><topic>Kinetics</topic><topic>Lyases</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Protein Conformation</topic><topic>Salmonella typhimurium</topic><topic>Salmonella typhimurium - enzymology</topic><topic>site-directed mutagenesis</topic><topic>Spectrophotometry, Ultraviolet</topic><topic>tryptophan synthase</topic><topic>Tryptophan Synthase - chemistry</topic><topic>Tryptophan Synthase - genetics</topic><topic>Tryptophan Synthase - metabolism</topic><topic>U.V. spectroscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>BRZOVIC, P. S</creatorcontrib><creatorcontrib>SAWA, Y</creatorcontrib><creatorcontrib>HYDE, C. C</creatorcontrib><creatorcontrib>MILES, E. W</creatorcontrib><creatorcontrib>DUNN, M. F</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><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>BRZOVIC, P. S</au><au>SAWA, Y</au><au>HYDE, C. C</au><au>MILES, E. W</au><au>DUNN, M. F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1992-06-25</date><risdate>1992</risdate><volume>267</volume><issue>18</issue><spage>13028</spage><epage>13038</epage><pages>13028-13038</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations
in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit
active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe
to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for
Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady
state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction.
However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are
observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for
each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution
into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., BrzoviÄ, P., Drewe, W.F.,
Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are
part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced
inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed"
structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of
indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence
of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open
to the closed form.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>1618800</pmid><doi>10.1016/S0021-9258(18)42377-0</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1992-06, Vol.267 (18), p.13028-13038 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_16389150 |
| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Allosteric Regulation alpha subunit Analytical, structural and metabolic biochemistry Base Sequence beta subunit Binding Sites biological activity Biological and medical sciences DNA Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Indoles - metabolism Kinetics Lyases Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Protein Conformation Salmonella typhimurium Salmonella typhimurium - enzymology site-directed mutagenesis Spectrophotometry, Ultraviolet tryptophan synthase Tryptophan Synthase - chemistry Tryptophan Synthase - genetics Tryptophan Synthase - metabolism U.V. spectroscopy |
| title | Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex |
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