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.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
J J
JJ
Hedberg
P
P
Strömberg
J O
JO
Höög
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