FIG. 5.

Substrate kinetics of the reaction catalyzed by L protein as a function of lipoylated H1, H2, and lipoamide as substrates. Purified recombinant proteins were used in reactions conducted at 25°C in a buffer containing 40 mM Tris, pH 7.4, 0.1 mM EDTA, 100 μM L protein, and 0.5 μM to 30 μM H1 protein (A), 0.5 μM to 30 μM H2 protein (B), or 0.01 mM to 1.0 mM lipoamide (C). The reaction was initiated by adding 2 mM Nbs₂. L protein activity was measured spectrophotometrically at 415 nm and expressed as nmol of Nbs formed/s.