Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Figures & data

Figure 1 Effect of cibacron blue on the initial velocities of 3-HBA-6-hydroxylase; a) activity measured by fluorimetric assay. b) activity measured by polarographic method.

Figure 2 Double reciprocal plots for demonstrating the nature of inhibition of 3-HBA-6-hydroxylase activity by cibacron blue with respect to 3-HBA.

Figure 3 Profile of quenching of intrinsic fluorescence (emission maximum at 335 nm) of 3-HBA-6- hydroxylase by cibacron blue.

Figure 4 Double reciprocal plots for the observed changes in enzyme fluorescence with increasing concentrations of cibacron blue using native enzyme, FAD-enzyme, 3-HBA-enzyme and urea denatured enzyme.

Figure 5 Stinson and Holbrook plots for the quenching of enzyme fluorescence with increasing concentrations of cibacron blue using native enzyme, 3-HBA-enzyme, FAD-enzyme and urea denatured enzyme.

Table I.  Binding parameters, stoichiometry (r) and association constant (K_a), for the enzyme–cibacron blue interaction. The binding parameters were calculated from the equation: 1/(1−θ) K_a=[X_T]/θ−r[E_T].

System	r (Stoichiometry)	Ka (μM)
E (enzyme)+C.B. (cibacron blue)	0.86	4.5
E+3-HBA (60 μM)+C.B.	0.86	4.2
E+FAD+C.B.	1.03	2.4
E+Urea (1 M)+C.B.	0.80	0.44

Figure 6 Near-UV CD spectral profiles of 3-HBA-6-hydroxylase in the absence and presence of cibacron blue.