Table 3. Inhibition constants for B. anthracis lumazine synthase.

Experiments were conducted with recombinant BaLS. The assays were performed with the concentration of compound 2 (see Fig. 1 ▶) held constant, while the concentration of compound 1 was varied. Reaction mixtures contained 100 mM Tris–HCl pH 7.0, 100 mM NaCl and 5 mM DTT. For the competitive mechanism it is assumed that the inhibitor binds at the substrate (compound 1) binding site and inactivates the enzyme completely. This model is described by the following chemical equations: E + S ↔ ES (substrate binding at enzyme), K _s is the dissociation constant for this equilibrium; ES → E + P (conversion of the enzyme–substrate complex into enzyme and product), k _cat is the rate constant for this process; E + I ↔ EI (binding of inhibitor at the binding site for the substrate, i.e. compound 1), K _i is the inhibitor-dissociation constant for this equilibrium. For the partial mechanism it is assumed that the inhibitor binds at the binding site for compound 1 and inactivates the enzyme completely. In parallel it can bind at the enzyme away from the binding site for compound 1. In this case it can only partially inactivate the enzyme. This model is described by the following chemical equations: E + S ↔ ES (substrate binding by enzyme), K _s is the dissociation constant for this equilibrium; ES → E + P (conversion of the enzyme–substrate complex into enzyme and product), k _cat is the rate constant for this process; E + I ↔ EI (binding of inhibitor at the binding site for compound 1), K _i is the inhibitor-dissociation constant for this equilibrium; ES + I ↔ ESI (binding of the inhibitor away from the binding site for compound 1), K _is is the inhibitor-dissociation constant for this equilibrium; ESI → E + I + P [conversion of the triple complex (enzyme–substrate–inhibitor) into enzyme, inhibitor and product], k′_cat is the rate constant for this process. A numerical solution to the problem of minimizing a least-squares function over a space of reaction parameters was found using the Levenberg–Marquardt algorithm.

Compound	Mechanism	Ks (µM)	kcat (min−1)	Ki (µM)	Kis (µM)	k′cat (min−1)
JC33	Competitive	8.0 ± 0.5	0.60 ± 0.01	0.023 ± 0.006
JC72	Partial	8.5 ± 0.5	0.60 ± 0.01	0.43 ± 0.06	9.5 ± 3.5	0.28 ± 0.09
TS23	Partial	8.3 ± 0.4	0.60 ± 0.01	0.14 ± 0.03	0.85 ± 0.2	0.08 ± 0.01