Fig. 1.

Kinetics and coenzyme binding of GAPDH complexes. (A) View of GAPDH (NAD⁺)-CP12 with adjacent active sites. The CP12-bound (proximal) site makes extensive contacts with both NAD⁺ and GAPDH. CP12-Glu69 prevents clashes with NADPH binding to the distal site, as it would clash with the 2′-phosphate. (B) PRK activity for the ternary complex treated with either oxidized (GAPDH-CP12-PRK^ox; ■) or reduced DTT (GAPDH-CP12-PRK^red; □). All reactions were measured in triplicate and fitted using Michaelis-Menten kinetics. Rate of NADP⁺ reduction (C) and NAD⁺ reduction (D) were measured for 275 µM GAPDH (■), 275 µM GAPDH-CP12 (○), and 63 µM GAPDH-CP12-PRK (△) complexes at increasing concentrations of nucleotide substrate. The maximal velocity (V_max) with NADP⁺ is not inhibited in GAPDH-CP12, but is fully inhibited in GAPDH-CP12-PRK. In contrast, the V_max with NAD⁺ is equally inhibited by GAPDH-CP12 and GAPDH-CP12-PRK.