Linked Life Data

7046786

Source:http://linkedlifedata.com/resource/pubmed/id/7046786

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
1982-9-10
pubmed:abstractText
Kinetics of complex formation between phenylalanyl-tRNA synthetase and Phe-tRNAPhe have been measured by the stopped-flow technique. Either the protein intrinsic fluorescence or the fluorescence of the added indicator 6-(p-toluidinyl)naphthalene-2-sulfonate was observed. Identical results were obtained with each method. Acyl-tRNAs with variable structures of the acyl and tRNA moieties were examined. Kinetics were measured as a function of pH and at different ionic strengths. Kinetic constants were compared with those of enzymatic phenylalanylation of tRNAPhe. The results are as follows. (1) Phe-tRNAPhe binds to phenylalanyl-tRNA synthetase in two, mutually exclusive types of complexes, one at the tRNA-specific binding site of the enzyme and the other in a region which involves the Phe-specific binding site of the enzyme [Holler, E. (1980) Biochemistry 19, 1397-1402]. The Phe site directed association includes a conformational change of the complex that is rate limiting. (2) The conformational change and catalytic tRNA aminoacylation follow similar values of rate constants irrespectively of pH and ionic strength. It is concluded that aminoacylation is limited by the kinetics of a conformational change of the nascent enzyme-Phe-tRNAPhe complex. (3) The nature of Phe site directed binding was probed by variation of the structure of Phe-tRNAPhe. Both the acyl and the tRNA moieties are recognized by the enzyme. Of the acyl moiety, only the phenyl ring but not the amino group is essential for binding. The amino group can be acetylated or replaced by a hydroxyl group. Protonation of the amino group results in loss of Phe site directed binding. It gives a pKa = 6.9, which is close to that for protonation of a phenylalanine ester. (4) Rate constants were only slightly affected by addition of 200 mM NaCl at pH 7.5, indicating that the contribution by electrostatic forces was probably minimal. Mg2+ ions were essential for Phe site directed binding. Complexation of enzyme, Phe-tRNAPhe, and Mg2+ either was random or was at preequilibrium with the conformational change. (5) Binding of Phe-tRNAPhe at the tRNA-specific site of the enzyme was studied in the presence of in situ synthesized phenylalanyl adenylate. The reaction was bimolecular with rate constants of 50 microM-1 s-1 and 15 s-1 for association and dissociation, respectively.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
21
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2460-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1982
pubmed:articleTitle
Kinetics of acyl transfer ribonucleic acid complexes of Escherichia coli phenylalanyl-tRNA synthetase. A conformational change is rate limiting in catalysis.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't