Section 69
Chapter 68,634

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

Lin, S.X.; Baltzinger, M.; Remy, P.

Biochemistry 22(3): 681-689


ISSN/ISBN: 0006-2960
PMID: 6340722
DOI: 10.1021/bi00272a024
Accession: 068633481

The discrimination by yeast phenylalanyl-tRNA synthetase between phenylalanine and tyrosine was studied with highly purified tyrosine. Commercial grade tyrosine contains enough contaminating phenylalanine (.simeq. 0.5% molar ratio) to perturb significantly the study of tyrosine misactivation. Highly purified tyrosine (9-fold recrystallized, containing < 5 .times. 10-5 mol/mol contaminating phenylalanine) was used. Tyrosine is indeed misactivated by phenylalanyl-tRNA synthetase, as shown by ATP-PPi exchange reaction. The maximal velocity of ATP-PPi exchange in the presence of tyrosine is < 20% of that occurring in the presence of phenylalanine. Titration experiments of phenylalanyl-tRNA synthetase by tyrosine in the presence of ATP or by chemically synthesized tyrosyladenylate, in the presence of 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a fluorescent reporter probe, reveal that phenylalanyl-tRNA synthetase has a rather poor affinity for tyrosyladenylate. This was confirmed by stopped-flow measurement of the kinetic association and dissociation constants. The forward rate constant for tyrosyladenylate was much lower than that for the cognate amino acid phenylalanine. The pyrophosphorolysis rate constant was at least equal to or even higher than that for phenylalanine. The discrimination between tyrosine and phenylalanine by phenylalanyl-tRNA synthetase appears to correspond to the model proposed by Hopfield (1974).

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