Enzymatic mechanism of low-activity mouse alcohol dehydrogenase 2.
ADH2 is a member of one of the six classes of mammalian alcohol dehydrogenases, which catalyze the reversible oxidation of alcohols using NAD(+) as a cofactor. Within the ADH2 class, the rodent enzymes form a subgroup that exhibits low catalytic activity with all substrates that were examined, as compared to other groups, such as human ADH2. The low activity can be ascribed to the rigid nature of the proline residue at position 47 as the activity can be increased by approximately 100-fold by substituting Pro47 with either His (as found in human ADH2), Ala, or Gln. Mouse ADH2 follows an ordered bi-bi mechanism, and hydride transfer is rate-limiting for oxidation of benzyl alcohols catalyzed by the mutated and wild-type enzymes. Structural studies suggest that the mouse enzyme with His47 has a more closed active site, as compared to the enzyme with Pro47, and hydride transfer can be more efficient. Oxidation of benzyl alcohol catalyzed by all forms of the enzyme is strongly pH dependent, with pK values in the range of 8.1-9.3 for turnover numbers and catalytic efficiency. These pK values probably correspond to the ionization of the zinc-bound water or alcohol. The pK values are not lowered by the Pro47 to His substitution, suggesting that His47 does not act as a catalytic base in the deprotonation of the zinc ligand.
Strömberg
Patrik
P
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Svensson
Stefan
S
Berst
Kristine B
KB
Plapp
Bryce V
BV
Höög
Jan-Olov
JO
D000409:Alanine / Q000235:genetics; D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019905:Benzyl Alcohol / Q000737:chemistry; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D011392:Proline; D000426:Alcohol Dehydrogenase; C105552:alcohol dehydrogenase IV; D019905:Benzyl Alcohol; D000409:Alanine