X-ray Structure of Human β3β3 Alcohol Dehydrogenase

• Published in: The Journal of biological chemistry 1996-07, Vol.271 (29), p.17057
• Main Authors: Gerard J. Davis, William F. Bosron, Carol L. Stone, Kwabena Owusu-Dekyi, Thomas D. Hurley
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.29.17057

The three-dimensional structure of the human β 3 β 3 dimeric alcohol dehydrogenase (β 3 ) was determined to 2.4-Å resolution. β 3 was crystallized as a ternary complex with the coenzyme NAD + and the competitive inhibitor 4-iodopyrazole. β 3 is a polymorphic variant at ADH2 that differs from β 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 β 3 cannot form this interaction. The three-dimensional structures of β 3 and β 1 are virtually identical, with the exception that Cys-369 and two water molecules in β 3 occupy the position of Arg-369 in β 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, β 3 differs from β 1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of β 3 for NAD + and NADH are 350-fold and 4000-fold higher, respectively, than those for β 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 β 3 isoenzyme destabilizes the interaction between coenzyme and β 3 alcohol dehydrogenase.