Chemical Modification of the Phosphoenolpyruvate Carboxylase from Maize Leaves and Its Conformation in Isotropic Solution. Studies via Triplet Lifetime and Rotational Diffusion Using Eosin Isothiocyanate as Label

C.S. ANDREO, A.A. IGLESIAS, F.E. PODESTÁ, R. WAGNER

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

Año: 1986 vol. 870 p. 292 - 301

0304-4165

Phosphoenolpyruvate carboxylase from maize leaves was covalently labeled with the triplet probe, eosin-5-isothiocyanate. The effect of chemical modification on the enzyme activity was investigated. The enzymeboundtriplet probe was used to measure the rotational diffusion of the enzyme in isotropic solution.Modification of the enzyme with eosin isothiocyanate resulted in a loss of enzymatic activity in a time- andconcentration-dependent manner. Complete inactivation could be correlated with the incorporation of 4 molof eosin isothiocyanate/moi of enzyme. However, complete protection against inactivation could be obtainedby phosphoenoipyruvate plus Mg 2+. The labeled enzyme revealed a rotational correlation time of 1 ps inaqueous buffer at 20°C, either in the absence or in the presence of all substrates. When HCO 3- and Mg 2+ orphosphoeno/pyruvate and Mg 2÷ were present only, a rotational correlation time of 2.1 ps was obtained.These results indicate the presence of four essential e-NH 2 groups in the enzyme, which are modulated byphosphoenoipyruvate plus Mg 2+, either in their location within the protein or/and in their apparent pK(reactivity to eosin isothiocyanate). From the rotational correlation times obtained, the hydrodynamicstructure of the enzyme in the absence or presence of all substrates could be described best by a tetrahedronarrangement of four monomeric subunits each with an apparent molecular weight of 100 000.