Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.
The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a remarkably old isozyme duplication or, more probably, discordant accumulation of residue exchanges with greater speed of evolution for one of the subunits (h chain) than is typical for the slowly evolving class III alcohol dehydrogenase.
Danielsson
O
O
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Shafqat
J
J
Estonius
M
M
el-Ahmad
M
M
Jörnvall
H
H
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002175:Candida; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D049831:Octopodiformes; D018532:Pisum sativum; D010542:Peromyscus; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae
D007527:Isoenzymes; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D005978:Glutathione