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http://hdl.handle.net/10261/111051
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dc.contributor.author | Rosell, Albert | - |
dc.contributor.author | Valencia, Eva | - |
dc.contributor.author | Ochoa, Wendy F. | - |
dc.contributor.author | Fita, Ignacio | - |
dc.contributor.author | Parés, Xavier | - |
dc.contributor.author | Farrés, Jaume | - |
dc.date.accessioned | 2015-02-23T13:47:11Z | - |
dc.date.available | 2015-02-23T13:47:11Z | - |
dc.date.issued | 2003-10-17 | - |
dc.identifier | doi: 10.1074/jbc.M307384200 | - |
dc.identifier | issn: 0021-9258 | - |
dc.identifier.citation | Journal of Biological Chemistry 278(42): 40573-40580 (2003) | - |
dc.identifier.uri | http://hdl.handle.net/10261/111051 | - |
dc.description.abstract | Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly 223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/K mNADPH = 760 mM-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133,330 mM-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155,000 mM-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family. | - |
dc.description.sponsorship | This work was supported by grants from the Dirección General de Investigación Científica: BMC2000-0132 (to J. F.), BMC2002-02659 (to X. P.), and BIO2002-04419-C02-01 (to I. F.) | - |
dc.publisher | American Society for Biochemistry and Molecular Biology | - |
dc.rights | closedAccess | - |
dc.title | Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase | - |
dc.type | artículo | - |
dc.identifier.doi | 10.1074/jbc.M307384200 | - |
dc.relation.publisherversion | http://dx.doi.org/10.1074/jbc.M307384200 | - |
dc.date.updated | 2015-02-23T13:47:11Z | - |
dc.description.version | Peer Reviewed | - |
dc.language.rfc3066 | eng | - |
dc.type.coar | http://purl.org/coar/resource_type/c_6501 | es_ES |
item.openairetype | artículo | - |
item.grantfulltext | none | - |
item.cerifentitytype | Publications | - |
item.openairecristype | http://purl.org/coar/resource_type/c_18cf | - |
item.fulltext | No Fulltext | - |
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