A change in reaction specificity of sheep liver serine hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr.
Both serine hydroxymethyltransferase and aspartate aminotransferase belong to the alpha-class of pyridoxal-5'-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of serine hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing L-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and D-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 microM and 5. 2 x 10-3 s-1, respectively. This oxidation was not observed with either the wild type serine hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver serine hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.
Talwar
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Rao
N A
NA
Savithri
H S
HS
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D004583:Electrons; D016166:Free Radical Scavengers / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008353:Mannitol / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009243:NAD / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000378:metabolism
D016166:Free Radical Scavengers; D011994:Recombinant Proteins; D009243:NAD; D009580:Nitroblue Tetrazolium; D001224:Aspartic Acid; D008353:Mannitol; D013482:Superoxide Dismutase; D012696:Glycine Hydroxymethyltransferase; D001219:Aspartate Aminotransferases