Figure 5.

Elimination reaction catalyzed by serine racemase. (A) Serine racemase catalyzes stoichiometric production of pyruvate and ammonia, which corresponds to the amount of serine racemase consumption. The reaction media contained 50 mM Tris⋅HCl (pH 7.4), 10 mM l-serine O-sulfate, and 15 μM PLP. Reaction was started by addition of 10 μg/ml recombinant serine racemase. The values correspond to the amount of l-serine O-sulfate consumed and products formed after 30 min of reaction. (B) Production of pyruvate is inhibited by l-serine and aminooxyacetic acid. The conditions were the same as in A, except that reaction media contained 0.2 mM NADH, 1 μg/ml lactate dehydrogenase, and recombinant serine racemase (2 μg/ml final concentration). Pyruvate production was continuously monitored by the decrease of absorbance at 340 nm. Control (○), 20 mM l-serine (●), or 1 mM AOAA (▴). (C) Noncompetitive inhibition of pyruvate production by l-serine. The reaction was as in A, except that media contained either no (○) or 5 mM (●) l-serine. (Inset) A double-reciprocal plot of the substrate curves. The values are representative of three to five different experiments with different enzyme preparations.