Phospholipase A2 complexes with gadolinium(III) and interaction of the enzyme-metal ion complex with monomeric and micellar alkylphosphorylcholines. Water proton nuclear magnetic relaxation studies

• Published in: Biochemistry (Easton) 1976-06, Vol.15 (11), p.2268-2274
• Main Authors: Hershberg, Robert D, Reed, George H, Slotboom, Arend J, DeHaas, Gerard H
• Format: Article
• Language: English
• Subjects: Animals; Colloids; Gadolinium; Kinetics; Macromolecular Substances; Magnetic Resonance Spectroscopy; Mathematics; Micelles; Pancreas - enzymology; Phospholipases; Phospholipids; Protein Binding; Protein Conformation; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00656a004

Gadolinium (III) binds competitively with calcium(II) to porcine pancreatic phospholipase A2 (EC 3.1.1.4) and its zymogen. The enzyme-Gd3+ complex exhibits 4% of the hydrolytic activity of the corresponding Ca2+ complex toward a dispersion of dioctanoyllecithin. Dissociation constants for the Gd3+ complex of enzyme and proenzyme were evaluated from water proton relaxation rate (PRR) titrations. At pH 5.8, the dissociation constants for the Gd3+ complexes of enzyme and zymogen are 0.50 and 0.18 mM, respectively. Dissociation constants for the complexes of enzyme with Ca2+, Eu3+, and Tb3+ were evaluated in PRR titrations by competition of these cations with Gd3+ binding. PRR enhancement factors for the Gd3+ complexes of enzyme and proenzyme are 16.4 and 5.8, respectively, at 22 degrees C and 24.3 MHz. Binding of a homologous series of n-alkylphosphorylcholines to the enzyme-Gd3+ complex was investigated through the influence of monomeric and micellar forms of these amphiphiles on the PRR enhancement factor for the enzyme-bound Gd3+. Separate monomer and micelle binding regions were observed in titrations using n-alkylphosphorylcholines with critical micelle concentrations ranging from 15 muM to 13 mM. In every case, the enhancement factors for the enzyme-Gd3+ complexes were significantly greater than that for the tenary complex, enzyme-Gd3+ -monomer. Morever, a synergism was observed in the binding of Gd3+ and micelles to the enzyme. The magnitudes of the PRR enhancement factors for the enzyme-Gd3+ complexes with micelles of n-alkylphosphorylcholines indicate that the bound Gd3+ is freely accessible to the bulk solvent. These results suggest a model for the enzyme-micelle complex in which the active site is spatially removed from the enzyme-micelle interface.