Lanthanide derivatives of phospholipase C from Bacillus cereus

• Published in: Biochimica et biophysica acta, Protein structure and molecular enzymology Vol. 744; no. 3; pp. 291 - 297
• Main Authors: El-Sayed, M.Y., Roberts, M.F.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.01.1983
• Subjects: 1H-NMR; B. cereus; Detergent-protein interaction; Lanthanide; lanthanide elements; Metal substitution; N.M.R; Phospholipase C; Bacillus cereus; Detergent-protein interaction; B. cereus; Lanthanide; 1H-NMR; Metal substitution; CTAB; Phospholipase C
• ISSN: 0167-4838; 1879-2588
• DOI: 10.1016/0167-4838(83)90202-9

Lanthanide derivatives of the zinc metalloprotein, phospholipase C ( Bacillus cereus), have been formed. Their reactivation from the half-apo enzyme (one zinc atom removed) was found to be very pH-dependent. The maximum reactivation activity obtained between pH 7–8 is approx. 40% of the native protein. Both the native enzyme and the Gd 3+-derivative show a plateau in activity toward dimyristoylphosphatidylcholine/Triton X-100 mixed micelles between pH 5.5–8.5 with an acid p Ka of 4.6 and a basic p Ka of 10.3. Substrate specificity is unchanged by lanthanide substitution: no activity is observed toward sphingomyelin or ether-linked lecithins. Both metalloenzymes show parallel ‘surface dilution’ kinetics towards dimyristoylphosphatidylcholine solubilized in mixed micelles with cetyltrimethylammonium bromide, zwittergent 3–14 and deoxycholate. However, the Gd 3+-derivative appears to be more strongly inhibited by Triton X-100 than the native enzyme. This may reflect a decreased K i for the nonionic detergent. Because of its similarity to the native protein, the Gd 3+-derivative is a very useful probe for NMR studies of protein interactions with lipids and detergents. For example, cetyltrimethylammonium bromide, a cationic detergent, binds tightly to the enzyme. The N-methyl protons are preferentially broadened over bulk methylene and the terminal methyl group when the detergent is bound to the protein, indicating the proximity of this part of the molecule to the substituted metal site on phospholipase C.