Linked Life Data

3305507

Source:http://linkedlifedata.com/resource/pubmed/id/3305507

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
1987-10-21
pubmed:abstractText
The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921; W. L. Finlayson and J. F. Kirsch, in preparation). Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate. Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525). This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F). Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ). The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F. In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT). Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT. Thus, Y70F dissociates coenzyme more readily than does WT. It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme. Tyr-70 does not function in an essential chemical step.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
262
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12403-5
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1987
pubmed:articleTitle
Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.