X-ray Structure of Human β3β3 Alcohol Dehydrogenase

• Published in: The Journal of biological chemistry 1996-07, Vol.271 (29), p.17057-17061
• Main Authors: Davis, Gerard J., Bosron, William F., Stone, Carol L., Owusu-Dekyi, Kwabena, Hurley, Thomas D.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.29.17057

The three-dimensional structure of the human β3β3 dimeric alcohol dehydrogenase (β3) was determined to 2.4-Å resolution. β3 was crystallized as a ternary complex with the coenzyme NAD+ and the competitive inhibitor 4-iodopyrazole. β3 is a polymorphic variant at ADH2 that differs from β1 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 β3 cannot form this interaction. The three-dimensional structures of β3 and β1 are virtually identical, with the exception that Cys-369 and two water molecules in β3 occupy the position of Arg-369 in β1. 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, β3 differs from β1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of β3 for NAD+ and NADH are 350-fold and 4000-fold higher, respectively, than those for β1. 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 β3 isoenzyme destabilizes the interaction between coenzyme and β3 alcohol dehydrogenase.