title: Comparative roles of histidine 51 in human beta 1 beta 1 and threonine 51 in pi pi alcohol dehydrogenases.
abstract: Histidine at position 51 of the class I beta 1 alcohol dehydrogenase (ADH) functions as a general base by indirectly abstracting a proton from the alcohol substrate through a hydrogen-bonded proton relay system. The human class II pi-ADH was reported to be polymorphic, having either Ser or Thr, but not His at position 51. It is unknown whether Ser or Thr51 have a catalytic role in ethanol oxidation with pi-ADH. Accordingly, we expressed and purified recombinant mutants of pi-ADH with Thr, Ser, and His at position 51. At pH 6.5, values for Vmax/Km for ethanol were 0.30, 0.10, and 0.09 min-1 mM-1 for pi 51Thr, pi 51Ser, and pi 51His ADH, respectively. Hence the effects of the substitutions were much less than the 11-fold decrease in Vmax/Km observed for beta 1-ADH when a neutral amino acid (Gln) was substituted for His51. Addition of a buffer base (400 mM glycylglycine) had little effect on Vmax/Km of recombinant pi 51Thr or pi 51Ser ADH, while it increased Vmax/Km for ethanol 7-fold for the beta (1)51 Gln ADH. We conclude that there is no evidence for Thr51 of pi-ADH participating in a proton relay similar to that seen in beta 1-ADH and that a base at position 51 may not be a universal requirement for a functional alcohol dehydrogenase with a moderate efficiency for ethanol oxidation at a physiological pH.
authors.affiliation: Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202-5122.
authors.forename: G J
authors.initials: GJ
authors.lastname: Davis
authors.forename: L G
authors.initials: LG
authors.lastname: Carr
authors.forename: T D
authors.initials: TD
authors.lastname: Hurley
authors.forename: T K
authors.initials: TK
authors.lastname: Li
authors.forename: W F
authors.initials: WF
authors.lastname: Bosron
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D014644:Genetic Variation; D005838:Genotype; D006639:Histidine; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D013912:Threonine
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D006639:Histidine; D000426:Alcohol Dehydrogenase