INVESTIGADORES
GONZALEZ Daniel Hector
artículos
Título:
Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study
Autor/es:
WAGNER, RICHARD; PODESTÁ, FLORENCIO E.; GONZALEZ, DANIEL H.; ANDREO, CARLOS S.
Revista:
EUROPEAN JOURNAL OF BIOCHEMISTRY
Editorial:
Elsevier
Referencias:
Año: 1988 vol. 173 p. 561 - 568
ISSN:
0014-2956
Resumen:
Phosphoenolpyruvate carboxylase,
purified from maize leaves, is rapidly inactivated by the fluorescence
probe dansyl chloride. The loss of activity can be ascribed to the
covalent modification of an R-NH2 group, presumably the epsilon-NH2
group of lysine. Analysis of the data by the statistical method of Tsou
[Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8
eight R-NH2 groups can be modified in the tetrameric form of the enzyme,
four of which are essential for catalytic activity. Essential groups
are modified about five times more rapidly than the non-essential ones.
The enzyme was completely protected against inactivation by Mg2+ plus
phosphoenolpyruvate and consequently binding of the modifier to the
essential groups is completely abolished. Hence the four essential
groups seemed to be located at or near the active site(s). One of the
four essential groups was modified with dansyl chloride and the other
three progressively with eosin isothiocyanate. In the doubly labeled
protein non-radiative single-singlet energy transfer between dansyl
chloride (donor) and eosin isothiocyanate (acceptor) was observed. The
low variance (+/- 5%) in the efficiency of energy transfer obtained at a
particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in
triplicate samples provided confidence that the measured transfer
efficiency may be interpreted as transfer between specific sites. The
distances calculated from the efficiency of resonance energy transfer
revealed two acceptor sites, equally separated, 4.8-5.1 nm from the
donor site and third site being 6.4 nm apart from the donor. Under
conditions where the tetrameric enzyme dissociates into the monomers, no
transfer of resonance energy between the protein-bound dansyl chloride
and eosin isothiocyanate was observed. Most likely the four essential
lysyl residues in the tetrameric enzyme are located in different
subunits of the enzyme, hence each of the subunits would contain a
substrate-binding site with one lysyl residue crucial for activity.