title: Involvement of threonine 234 in catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase.
abstract: There is a mobile loop in the tyrosyl-tRNA synthetase that contains the KMSKS signature sequence of class I aminoacyl-tRNA synthetases. As it has not been possible to determine the role of the mobile loop in catalysis from X-ray crystallographic studies, we are investigating its importance by a series of site-directed mutagenic and kinetic studies. Here we examine the role of threonine 234 (T234) in the catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase from Bacillus stearothermophilus. This residue is the carboxy-terminal residue in the signature sequence and is either a serine or threonine in eight of the ten class I aminoacyl-tRNA synthetases isolated from Escherichia coli. Kinetic analyses of tyrosyl adenylate formation in the mutant enzymes indicate that k3, the forward rate constant for the formation of tyrosyl adenylate, is reduced 500-fold on mutation of T234 to alanine. In contrast, mutation of T234 to serine results in only a 4-fold decrease in k3, suggesting that the loss of the hydroxyl group in the T234A mutant is responsible for its decreased reaction rate. Deletion of the hydroxyl group destabilizes the transition state for the formation of tyrosyl adenylate by 2.7 kcal/mol. The transition state is also destabilized by 1.4 kcal/mol on the mutation of K230 to alanine. The effects of mutation of both T234 and K230 to alanine are less than additive; there is a coupling energy of -1.3 kcal/mol in the transition state. The effects of mutating K230 and T234 to alanine are also nonadditive in the E.Tyr-AMP complex (coupling energy = -1.9 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
authors.forename: E A
authors.initials: EA
authors.lastname: First
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism*; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013912:Threonine; D014169:Transformation, Bacterial; D014443:Tyrosine / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002104:Cadmium; D013912:Threonine; D000249:Adenosine Monophosphate; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase; D008274:Magnesium