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 a lcohol dehydrogenase (beta(3)) was determined to 2.4-Angstrom resoluti on. beta(3) was crystallized as a ternary complex with the coenzyme NA D(+) and the competitive inhibitor 4-iodopyrazole. beta(3) is a polymo rphic variant at ADH2 that differs from beta(1) by a single amino acid substitution of Arg-369 --> Cys. The available x-ray structures of ma mmalian alcohol dehydrogenases show that the side chain of Arg-369 for ms 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 interacti on. The three-dimensional structures of beta(3) and beta(1) are virtua lly 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 o ccupy the same positions as two guanidino nitrogens of Arg-369. Hence, the number of hydrogen bonding interactions between the enzyme and NA D(H) are the same for both isoenzymes. However, beta(3) differs from b eta(1) by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain, The equilibrium dissociatio n 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) al cohol dehydrogenase.