Linked Life Data

14512428

Source:http://linkedlifedata.com/resource/pubmed/id/14512428

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
49
pubmed:dateCreated
2003-12-3
pubmed:abstractText
Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454-43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01-9% of wild type. The most striking change is in the pH-V(max) curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2-0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-V(max) profiles. V(max) is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
278
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
49323-31
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase.
pubmed:affiliation
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.