Linked Life Data

1572355

Source:http://linkedlifedata.com/resource/pubmed/id/1572355

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1992-6-2
pubmed:abstractText
Amino acid residue 48 in human alcohol dehydrogenase constitutes one of 21 residue differences between the common, adult-type isozyme subunits beta and gamma. It is at the inner part of the substrate pocket and has been ascribed a role in hydrogen-bond formation with both the substrate and coenzyme. In order to allow direct evaluation of its importance, Thr48 of a recombinant non-acetylated beta subunit was mutated to Ser (as in the gamma subunit) or Ala (as in no native form, and not allowing side-chain hydrogen bonds), and the proteins were expressed in Escherichia coli. The two non-acetylated recombinant proteins, the beta 48T form and the mutant beta 48S, gave enzymatically active enzymes with indistinguishable specific activities towards ethanol, whereas the mutant beta 48A showed no enzymatic activity. The most striking differences between dimers with the beta subunit and the beta 48S subunit (both non-acetylated) were observed with cyclohexanol, hydroxysteroids, methanol and ethanol. With cyclohexanol, the Km was lowered from 11 mM to 280 microM, and the kcat/Km ratio, although still less than that for the gamma gamma isozyme, was increased 80-fold. Similarly, beta 48S could use 3 beta-hydroxy-5 beta-androstan-17-one as substrate, like gamma gamma, although again with a catalytic efficiency much less than that for the gamma gamma isozyme. Furthermore, testosterone inhibited beta 48S to 50% at a concentration of 100 microM, whereas the beta beta form was not inhibited. All these results show that residue 48 is responsible for a large part of the differences between the two isozymes beta beta and gamma gamma of human class-I alcohol dehydrogenase. The form with the inactive beta 48A subunit was possible to purify by AMP-Sepharose chromatography, suggesting the presence of a functional NAD-binding site. The enzymatic measurements, demonstrating a transition from one isozyme activity to that characteristic of another, confirmed that a side-chain hydroxyl in residue 48 is required for activity, and interpretation by computer modelling showed marked differences at the active site.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0014-2956
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
205
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
519-26
pubmed:dateRevised
2007-7-23
pubmed:meshHeading
pubmed-meshheading:1572355-Alcohol Dehydrogenase, pubmed-meshheading:1572355-Animals, pubmed-meshheading:1572355-Base Sequence, pubmed-meshheading:1572355-Binding Sites, pubmed-meshheading:1572355-Cloning, Molecular, pubmed-meshheading:1572355-Computer Graphics, pubmed-meshheading:1572355-Horses, pubmed-meshheading:1572355-Humans, pubmed-meshheading:1572355-Isoenzymes, pubmed-meshheading:1572355-Kinetics, pubmed-meshheading:1572355-Liver, pubmed-meshheading:1572355-Macromolecular Substances, pubmed-meshheading:1572355-Models, Molecular, pubmed-meshheading:1572355-Molecular Sequence Data, pubmed-meshheading:1572355-Mutagenesis, Site-Directed, pubmed-meshheading:1572355-Oligodeoxyribonucleotides, pubmed-meshheading:1572355-Protein Conformation, pubmed-meshheading:1572355-Recombinant Proteins, pubmed-meshheading:1572355-Testosterone
pubmed:year
1992
pubmed:articleTitle
A single-residue exchange gives human recombinant beta beta alcohol dehydrogenase gamma gamma isozyme properties.
pubmed:affiliation
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't