X-ray structure of human beta(3)beta(3) alcohol dehydrogenase - The contribution of ionic interactions to coenzyme binding

The three-dimensional structure of the human beta(3) beta(3) dimeric alcohol dehydrogenase (beta(3)) was determined to 2.4-Angstrom resolution. beta(3) was crystallized as a ternary complex with the coenzyme NAD(+) and the competitive inhibitor 4-iodopyrazole. beta(3) is a polymorphic variant at ADH2 that differs from beta(1) by a single amino acid substitution of Arg-369 --> Cys. The available x-ray structures of mammalian alcohol dehydrogenases show that the side chain of Arg-369 forms an ion pair with the NAD(H) pyrophosphate to stabilize the E NAD(H) complex. The Cys-369 side chain of beta(3) cannot form this interaction. The three-dimensional structures of beta(3) and beta(1) are virtually identical, with the exception that Cys-369 and two water molecules in beta(3) occupy the position of Arg-369 in beta(1) The two waters occupy the same positions as two guanidino nitrogens of Arg-369. Hence, the number of hydrogen bonding interactions between the enzyme and NAD(H) are the same for both isoenzymes. However, beta(3) differs from beta(1) by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain, The equilibrium dissociation constants of beta(3) for NAD(+) and NADH are 350-fold and 4000-fold higher, respectively, than those for beta(1). These changes correspond to binding free energy differences of 3.5 kcal/mol for NAD(+) and 4.9 kcal/mol for NADH. Thus, the Arg-369 --> Cys substitution of beta(3) isoenzyme destabilizes the interaction between coenzyme and beta(3) alcohol dehydrogenase.