X-ray structure of human beta3beta3 alcohol dehydrogenase. The contribution of ionic interactions to coenzyme binding.
The three-dimensional structure of the human beta3beta3 dimeric alcohol dehydrogenase (beta3) was determined to 2.4-A resolution. beta3 was crystallized as a ternary complex with the coenzyme NAD+ and the competitive inhibitor 4-iodopyrazole. beta3 is a polymorphic variant at ADH2 that differs from beta1 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 beta3 cannot form this interaction. The three-dimensional structures of beta3 and beta1 are virtually identical, with the exception that Cys-369 and two water molecules in beta3 occupy the position of Arg-369 in beta1. 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, beta3 differs from beta1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of beta3 for NAD+ and NADH are 350-fold and 4000-fold higher, respectively, than those for beta1. 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 beta3 isoenzyme destabilizes the interaction between coenzyme and beta3 alcohol dehydrogenase.
Davis
G J
GJ
Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Bosron
W F
WF
Stone
C L
CL
Owusu-Dekyi
K
K
Hurley
T D
TD
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D003545:Cysteine; D018076:DNA, Complementary; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009243:NAD; D001120:Arginine; D000426:Alcohol Dehydrogenase; D003545:Cysteine