| pubmed-article:12163495 | rdf:type | pubmed:Citation | lld:pubmed |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C0038409 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C0002520 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C0017857 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C1414968 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C0038592 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C1314939 | lld:lifeskim |
| pubmed-article:12163495 | lifeskim:mentions | umls-concept:C1880022 | lld:lifeskim |
| pubmed-article:12163495 | pubmed:issue | 42 | lld:pubmed |
| pubmed-article:12163495 | pubmed:dateCreated | 2002-10-15 | lld:pubmed |
| pubmed-article:12163495 | pubmed:abstractText | In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k(cat) and K(m) for d-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K(m), on k(cat), or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity for d-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 10(5). With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and the K(m) of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficiently d-G3P relative to Cys-302 within the ternary complex GAPN.NADP.d-G3P. | lld:pubmed |
| pubmed-article:12163495 | pubmed:language | eng | lld:pubmed |
| pubmed-article:12163495 | pubmed:journal | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:citationSubset | IM | lld:pubmed |
| pubmed-article:12163495 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:chemical | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:12163495 | pubmed:status | MEDLINE | lld:pubmed |
| pubmed-article:12163495 | pubmed:month | Oct | lld:pubmed |
| pubmed-article:12163495 | pubmed:issn | 0021-9258 | lld:pubmed |
| pubmed-article:12163495 | pubmed:author | pubmed-author:BranlantGuyG | lld:pubmed |
| pubmed-article:12163495 | pubmed:author | pubmed-author:MarchalStepha... | lld:pubmed |
| pubmed-article:12163495 | pubmed:issnType | Print | lld:pubmed |
| pubmed-article:12163495 | pubmed:day | 18 | lld:pubmed |
| pubmed-article:12163495 | pubmed:volume | 277 | lld:pubmed |
| pubmed-article:12163495 | pubmed:owner | NLM | lld:pubmed |
| pubmed-article:12163495 | pubmed:authorsComplete | Y | lld:pubmed |
| pubmed-article:12163495 | pubmed:pagination | 39235-42 | lld:pubmed |
| pubmed-article:12163495 | pubmed:dateRevised | 2006-11-15 | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
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| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:meshHeading | pubmed-meshheading:12163495... | lld:pubmed |
| pubmed-article:12163495 | pubmed:year | 2002 | lld:pubmed |
| pubmed-article:12163495 | pubmed:articleTitle | Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans. | lld:pubmed |
| pubmed-article:12163495 | pubmed:affiliation | Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR CNRS-UHP 7567, Université Henri Poincaré Nancy 1, Faculté des Sciences, BP 239, F-54506 Vandoeuvre-lès-Nancy Cédex, France. | lld:pubmed |
| pubmed-article:12163495 | pubmed:publicationType | Journal Article | lld:pubmed |
| pubmed-article:12163495 | pubmed:publicationType | Research Support, Non-U.S. Gov't | lld:pubmed |
| http://linkedlifedata.com/r... | pubmed:referesTo | pubmed-article:12163495 | lld:pubmed |
