Engineering of the Nonspecific Phospholipase C from Bacillus cereus:  Replacement of Glutamic Acid-4 by Alanine Results in Loss of Interfacial Catalysis and Enhanced Phosphomonoesterase Activity

Abstract

The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.

Phospholipases Used in Lipid Transformations

“…As concluded from temperature factors for the protein backbone and structural studies of complexes between PLC and various inhibitors, substrate analogs and reaction products, the molecule contains a highly stable inner core and is hardly influenced by ligand binding (reviewed in Hough and Hansen, 1994). Studies by site-directed mutagenesis showed that Glu4 belonging to a highly flexible region flanking the active site is important for substrate binding, but not for catalysis (Tan and Roberts, 1998). These regions form a nonpolar surface and may influence substrate -enzyme interactions prior to binding in the active site.…”

Phosphatidylcholine-Preferring Phospholipase C from B. cereus. Function, Structure, and Mechanism

“…13) and similar to that of wild-type. In the other inves-tigation, the E4A mutant was observed to have greater phosphomonoesterase activity than wild-type PLC Bc [94].…”

“…There have been two independent mutagenesis studies that have been directed toward probing the role of E4 in the PLC Bc reaction [36,94]. In the first of these, the kinetic parameters k cat and K m of the E4L, E4D, and E4Q mutants, which each gave CD spectra similar to wild-type, were determined by the choline quantitation method [33], and these mutants were found to retain 6-60% of the catalytic efficiency (i.e., k cat /K m ) of wild-type [36,64].…”

Probing Polyethylene Glycol–Phospholipid Membrane Interactions Using Enzymes

“…Interfacial activation is a structural property of an enzyme; hence it is conceivable that the differences seen between PLA 2 and PLC activities may reflect their responses to altered membrane interfaces in PEGs (24,27). The effects of PEGs were different when PEG was conjugated to the lipid.…”