Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements

• Published in: Archives of biochemistry and biophysics Vol. 281; no. 2; pp. 324 - 329
• Main Authors: Wu, M.X. (University of California, Riverside, CA), Meyer, C.R, Willeford, K.O, Weeding, R.T
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier 01.09.1990
• Subjects: ACTIVADOR ENZIMATICO; ACTIVATEUR D'ENZYME; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carboxy-Lyases - analysis; ENZYME ACTIVATORS; ENZYME INHIBITORS; Enzyme Stability; ENZYME SUBUNITS; Enzymes and enzyme inhibitors; FOSFENOLPIRUVATO CARBOXILASA; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; INTERACTIONS; Ligases; Light; Macromolecular Substances; MOLECULAR WEIGHT; PEPTIDE; PEPTIDES; PEPTIDOS; PESO MOLECULAR; PHOSPHOENOLPYRUVATE CARBOXYLASE; Phosphoenolpyruvate Carboxylase - analysis; POIDS MOLECULAIRE; POLYPEPTIDES; Protein Conformation; PROTEIN STRUCTURE; Scattering, Radiation; ZEA MAYS; Zea mays - enzymology; Monocotyledones; Vegetals; Oligomerization; Stability; Zea mays; Chemical precipitation; Molecular weight determination; Gel electrophoresis; Enzyme; HPLC chromatography; Molecular dissociation; Aggregation; Characterization; Enzymatic activity; Gramineae; Angiospermae; Gel filtration; Spermatophyta; Phosphoenolpyruvate carboxylase; Ultraviolet visible spectrometry
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(90)90451-4

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.