Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis.
By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.
Brisdelli
Fabrizia
F
Department of Biomedical and Technological Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100 L'Aquila, Italy.
Saliola
Michele
M
Pascarella
Stefano
S
Luzi
Carla
C
Franceschini
Nicola
N
Falcone
Claudio
C
Martini
Filippo
F
Bozzi
Argante
A
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D005656:Fungal Proteins; D007527:Isoenzymes; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases