Table 1.

Relative activities (Rel. Act.),^a diC₇PC activation of cIP hydrolysis, apparent K_d (determined by non-FCS methods) for pure PC SUVs, and kinetic defects of B. thuringiensis PI-PLC variants extracted from published work (13–15, 24).

PI-PLC	Rel. Act. (PI/diC7PC)	PC activation of cIP hydrolysisb$\frac{{\mathrm{v}}_{cIP}(+di{C}_{7}PC)}{{\mathrm{v}}_{cIP}}$	Kd (mM), PC SUVs (non-FCS)	comment / kinetic defect
N168C	1.00	22	0.064±0.010 c,d	both enzyme activities are enhanced by PC binding; synergistic effect of PG/PC on Kd for SUVs determined by FCS
P42G	0.70	5.8	0.77±0.30 c	PC activation of cIP hydrolysis is impaired, while PI cleavage is close to wildtype; PC binding is impaired
K44A	0.98	33	0.087±0.005 d	PI cleavage in PI/diC7PC micelles and PC activation of cIP hydrolysis like wildtype; binding to PC SUVs like wildtype
K44E	0.44	4.5	0.5±0.2 d	PI cleavage in PI/diC7PC micelles and PC activation of cIP hydrolysis are significantly impaired; binding to PC vesicles is weaker
Y88A	2.92	28	0.94±0.05 e	PI cleavage in PI/diC7PC micelles is 2-fold higher than wildtype; PC activation of cIP hydrolysis comparable to wildtype; binding to PC SUVs is weakened
3YS f	0.36	2.9	0.5±0.2 d	PI cleavage in PI/diC7PC micelles is about 30% of wildtype; PC activation of cIP hydrolysis is significantly reduced; binding to PC SUVs is much weaker than wildtype

^aStandard assay systems include: 6 (or 8) mM PI dispersed in 24 (or 32) mM diC₇PC. The specific activity of wildtype enzyme is 1630 µmol min⁻¹ mg⁻¹ for PI/diC₇PC.

^bThe extent of PC activation of cIP hydrolysis is measured as the ratio of the specific activity, v_cIP, in the presence of 5 mM diC₇PC over v_cIP of the enzyme towards cIP alone. For 20 mM cIP in the absence of diC₇PC v_cIP was typically 2.8 µmol min⁻¹ mg⁻¹.

^cMeasured by intrinsic fluorescence of PI-PLC when titrated with pure SUVs (13).

^dMeasured by filtration assay, which briefly concentrates the sample (24).

^eUnpublished results from S. Guo, Boston College.

^fData from (14).