Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

Abstract

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 μM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (K_D = 0.29 μM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH · NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (K_D = 1.4 μM) and that determined for the soluble complex (K_D = 2.4 μM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 μM vs 22 μM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 μM and 43 μM, respectively). These results confirm the proposed mechanism of enhancement of the catalytic rate by ADP binding via a weakening of coenzyme binding in the active ternary complex. A method is also presented for evaluation of very weak interactions within ternary systems. This method yields dissociation constants for the GDH · glutamate complex of 52 mM and 2.8 mM with and without the regulatory site occupied by ADP, respectively. Hence the analytical affinity chromatography allows determination of affinity constants that range over 5 orders of magnitude.