Kinetic evidence for half-of-the-sites reactivity in tRNA/sup Trp/ aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

Journal Article · Tue Nov 04 04:00:00 EST 1986 · Biochemistry; (United States)

OSTI ID:6439483

Trezeguet, V; Merle, M; Gandar, J C; Labouesse, B

The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flowed method. The transfer of tryptophan to tRNA/sup Trp/ was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-(/sup 14/C)tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-(/sup 14/C)tryptophan were used. The authors measured either the formation of a single labeled aminoacyl-tRNA/sup Trp/ per enzyme subunit or the turnover of labeled aminoacyl-tRNA/sup Trp/ synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute half-of-the-sites reactivity); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNA/sup Trp/ under each labeling conditions were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate if aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.

• Research Organization: CNRA, Bordeaux, France
• OSTI ID: 6439483
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 25:22; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACYLATION
ALKALINE EARTH METAL COMPOUNDS
AMINO ACIDS
ANIMALS
AROMATICS
ATP
AZAARENES
AZOLES
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CATTLE
CHARGED PARTICLES
CHEMICAL REACTIONS
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENDOCRINE GLANDS
ENZYMES
GLANDS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INDOLES
IONS
KINETICS
LABELLED COMPOUNDS
LIGASES
MAGNESIUM COMPOUNDS
MAMMALS
MATHEMATICAL MODELS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PANCREAS
PYRROLES
REACTION KINETICS
RNA
RUMINANTS
TRANSFER RNA
TRYPTOPHAN
VERTEBRATES