Isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids. cDNA cloning, expression, and comparison of active sites.
Horse liver alcohol dehydrogenase occurs as isoenzymes: E is active on ethanol but not steroids; S is active on ethanol and steroids. The cDNAs for these isoenzymes were cloned; both were 1.8-kilobase long and contained complete coding sequences. Both enzymes were expressed in Escherichia coli, and the purified proteins had properties similar to those of the natural enzymes. The amino acid sequence deduced from the open reading frame of the E-type cDNA agreed with the amino acid sequence of the E isoenzyme determined by protein sequencing and x-ray crystallography. When compared with the E-type cDNA, the coding region of the S-type cDNA contains 24 substitutions and 3 deletions, giving rise to an amino acid sequence for the S. isoenzyme that differs from that of the E isoenzyme at 10 positions: nine conservative substitutions and one deletion, of Asp-115. These changes can be accommodated in the three-dimensional structure of the E isoenzyme, and models of the E and S isoenzymes complexed with a 3 beta-hydroxy-5 beta-steroid were built. The modeling shows that Leu-116 apparently sterically hinders binding of steroids in the E isoenzyme, and deletion in the S isoenzyme of Asp-115 moves Leu-116 and relieves the hindrance. The human gamma and rat liver enzymes are also active on steroids, but they have a different constellation of amino acid residues in the substrate pocket. Thus, there are multiple bases for the activity on steroids.
Park
D H
DH
Department of Biochemistry, University of Iowa, Iowa City 52242.
Plapp
B V
BV
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006736:Horses; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014462:Ultrafiltration
D000438:Alcohols; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D000426:Alcohol Dehydrogenase