Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2.
The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.
Strömberg
P
P
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Svensson
S
S
Hedberg
J J
JJ
Nordling
E
E
Höög
J O
JO
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D005091:Exons / Q000235:genetics; D005787:Gene Frequency; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D000426:Alcohol Dehydrogenase