Front Matter

cluster on the inner surface of a deep cleft in the molecule. The metal cluster is a part

of the active site and attracts the phosphate group of the substrate molecule. Accord-

ing to molecular modeling (Byberg et al., 1992), the substrate is bound via co-ordi-

nation of two phosphate oxygens to the three metal ions and co-ordination of the ester

carbonyl from the C(2) fatty acid to one Zn2+. In analogy with other phosphodies-

terases, the reaction is suggested to proceed by an in-line attack of an activated water

molecule on the phosphodiester, followed by collapse of the resulting penta-coor-

dinate intermediate to give 1,2-diacylglycerol and the phosphorylated head group

with inversion of stereochemistry at phosphorus. From studies by site-directed mu-

tagenesis it has been concluded that Asp55 likely serves as the general base in the

catalysis (Martin and Hergenrother, 1998a).

Although the enzyme does not contain disulfide bonds, it is extremely stable, even

in the presence of 8 M urea at 40 8 C (Little, 1978), but loses its stability when the

Zn2+ions are removed. In guanidine hydrochloride the enzyme can be unfolded and

refolded reversibly by the removal and addition of Zn2+(Little and Johansen, 1979).

As concluded from temperature factors for the protein backbone and structural stu-

dies of complexes between PLC and various inhibitors, substrate analogs and reac-

tion products, the molecule contains a highly stable inner core and is hardly influ-

enced by ligand binding (reviewed in Hough and Hansen, 1994). At the molecular

surface acidic, basic and neutral hydrophilic residues are distributed uniformly with

the exception of two loop regions adjacent to the active site. These regions form a

nonpolar surface and may influence substrate – enzyme interactions prior to binding

in the active site. 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).

PLC fromBacillus cereusis termed a broad-spectrum enzyme. In addition to its

preferred substrate PC, it also hydrolyzes phosphatidylserine (PS) or phosphatidyl-

ethanolamine (PE), but with lower activity. In analyzing the substrate specificity of

PLC, Massing and Eibl (1994) found that the negative charge at the phosphate group

must be balanced by a positive charge of the head group at suitable distance to fulfil

the substrate requirements of the enzyme. Good substrates for PLC fromB. cereus

have an ester bond at positionsn-2 and an ester or ether bond at thesn-1 position of

the glycerol, while an ether bond at thesn-2 position completely abolishes the ability

to act as substrate.

Interestingly, crystal structures of PI-PLCs do not resemble those of PC-specific

PLC or other phospholipases. They consist of a single domain folded as a (ab) 8 -

barrel and are metal-independent. The catalytic mechanism is similar to that of ri-

bonucleases (reviews in Katan, 1998; Griffith and Ryan, 1999).

12.3.3 Phospholipase D

The reason for the restricted information on the molecular properties of PLDs is

probably due to the difficulty in obtaining the enzyme in highly pure or crystallized

form. Highly purified PLDs from plants have been prepared from peanut seeds

(Heller et al., 1974), citrus callus (Witt et al., 1987), cabbage (Table 1), soybean

228 12 Phospholipases Used in Lipid Transformations