An attempt to transform class characteristics within the alcohol dehydrogenase family.
Human class I alcohol dehydrogenase was mutated at positions 57 and 115, exchanging for Asp and Arg respectively, in an attempt to introduce glutathione-dependent formaldehyde dehydrogenase characteristics. In addition, class III alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, was mutated at position 115, introducing Ser or Lys. The attempted class transformation was partly successful considering a higher affinity for 12-hydroxydodecanoate and a lower affinity for ethanol that was monitored for the class I mutant. However, the class I mutant displayed neither glutathione-dependent formaldehyde dehydrogenase activity nor fatty acid activation of alcohol oxidation. Interestingly, both class III mutants showed reduced activities for S-hydroxymethylglutathione and 12-hydroxydodecanoate through increased Km, values. Overall results show that it is not possible, by single point mutations, to completely transform enzyme characteristics between these two classes of alcohol dehydrogenase.
Hedberg
J J
JJ
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Strömberg
P
P
Höög
J O
JO
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001120:Arginine; D001665:Binding Sites; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D007930:Leucine; D008239:Lysine; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012694:Serine
D007850:Lauric Acids; D011994:Recombinant Proteins; C029469:S-hydroxymethylglutathione; D012694:Serine; C031263:12-hydroxydodecanoic acid; D001120:Arginine; D000426:Alcohol Dehydrogenase; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D005978:Glutathione; D007930:Leucine; D008239:Lysine