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269, and valine-270 are close to either the nicotinamide ring or the carboxamide moiety. In addition, the
face of the side chain of phenylalanine-94 is below the nicotinamide ring and its carboxamide group.
This latter interaction is unusual because phenylalanine-94 is between the two canonical glycine
residues in the βαβ fold, which is usually thought of as interacting mainly with the AMP part of the
cofactor. In 11β-HSD-2, there is an interesting configuration of amino acids with aromatic side chains
that are below the nicotinamide ring and which provide a hydrophobic cushion for NAD +.
E. Catalytic Site
Comparison of 11β-HSD-1 with homologs identifies tyrosine-183 and lysine-187 as being highly
conserved residues. Mutagenesis of these residues [40] and the homologous tyrosine and lysine in 17β-
HSD-1 and Drosophila alcohol dehydrogenase [44,45] shows that these residues are important for
catalytic function. The 3D model of 11β-HSD-1 presented in Figure 4 shows that tyrosine-183 is 3.6 Å
from the nicotinamide C4, where hydride transfer occurs. Similarly, in 11β-HSD-2, tyrosine-232 is 4 Å
from C4 on NAD +. Their positions support the notion that tyrosine is the catalytically active residue.
However, a problem with this model is that the pKa of tyrosine is about 10, which would make this
residue a poor nucleophile at neutral pH. To resolve this problem for the homologous tyrosine in
Drosophila alcohol dehydrogenase, Chen et al. [44] proposed that the pKa of tyrosine is lowered by a
nearby positively charged lysine. The 3D structure of the two 11β-HSDs shows that lysine-187 and
lysine-236 are close to the proposed catalytically active tyrosine residues, which supports the hypothesis
of Chen et al. [44].
F. Dimer Interface and the Catalytic Site
Most short-chain alcohol dehydrogenases are active as either dimers or tetramers. Analysis of rat
dihydropteridine reductase by Varughese et al. [33] indicates that the dimer interface consists of α helix
E and α helix F from each monomer arranged in a four α helix bundle, a structure in which the
hydrophobic surfaces on the helices form a core that yields very stable structure in a wide variety of
proteins [56–59]. A four-helix bundle also appears to stabilize S. hydrogenans 20β-hydroxysteroid
dehydrogenase, a tetrameric enzyme [34]. The α helix F contains the conserved tyrosine and lysine
residue, which adds a constraint to changes in the sequence of this helix. It has at least two functions:
stabilizing the dimer and orienting tyrosine and lysine and other residues for optimal interaction with
substrate and nucleotide cofactor.
The role of a specific site on the outer hydrophobic surface of α helix F in dimerization was suggested
recently when a Drosophila ADH mutant that does
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