Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP+) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP⁺. The origins of the difference in binding affinity to DHFR between NADPH and NADP⁺ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in ¹H and ¹⁵N chemical shift between NADPH and NADP⁺ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP⁺ compared with NADPH results partly from NADP⁺ having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP⁺ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.