Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

Abstract

The homodimeric Oriental 132132 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the .813f3 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the P2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the 81, form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the P13f1 and 82182 isozymes, including both a lower pH optimum and higher turnover number of 382132, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme.

Cloning and sequencing of cDNA encoding baboon liver alcohol dehydrogenase: evidence for a common ancestral lineage with the human alcohol dehydrogenase beta subunit and for class I ADH gene duplications predating primate radiation.

“…Remarkably, a sequence of 10 (Table 1) reveals a high degree of sequence identity (>93%) between these structures. Moreover, the human ADH-P and baboon ADH-,B cDNA sequences show the highest level of sequence identity (97.6%).…”

“…The nucleotide sequence of the cDNA insert of pBL14 was determined according to the strategy in Fig. 1 (49) 11 (28) iy~2 5 (69) 20 (49) 0 (0) 25 (61) Baboon (3 30 (73) 11 (28) 25 (61) 0 (0) The amino acid and nucleotide sequences of human ADH subunits (a, (31, and 9y) are from previous reports (3,(8)(9)(10)(11)(12)(13)(14)(15)(16)). …”

“…Human class I ADH isozymes exist as homo-and heterodimers of the a, 13, and fy subunits (ADH-a, -/3, and -y; ref. 7), for which structures are known at the protein and cDNA levels (3,(8)(9)(10)(11)(12)(13)(14)(15)(16). These class I subunits share positional identity at >90% of all positions.…”

Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

“…Atypical 02-subunits have been differently reported from livers of Caucasians and Asians (12-lern and f2-Oriental) (9,10), but both of these 2 types are now known to be structurally identical (11,12), differing from typical (1 subunits by a single His/Arg exchange at position 47. Another type of ADH, ADHIndianapolis, is probably derived from a further variant of the ADH2 locus (13).…”

“…son with the corresponding yeast ADH counterparts have provided structural explanations for the functional differences and have also illustrated the presence of parallel mutational exchanges (11).…”