title: Chemical modification and site-directed mutagenesis of Tyr36 of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.
abstract: 3-Isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was chemically modified with tetranitromethane which nitrated 1.5-2.0 Tyr residues per subunit. The nitration was biphasic and parallel to the loss of activity. The modified residue in the first phase was identified to be Tyr36, which is distantly located from the active site of the enzyme. The function of Tyr36 was investigated by site-specific replacement with Phe. The Michaelis constant for the substrate or co-enzyme was not altered by the replacement, whereas the catalytic constant decreased down to approximately 5%. X-ray analysis of the mutant enzyme revealed that Arg94 moved the largest distance among the active site residues, that is, the NH1 and NH2 of the guanidino group moved 1.11 and 1.32 A respectively. The results suggest that Arg94 is responsible for the enzyme catalysis.
authors.affiliation: Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
authors.forename: S
authors.initials: S
authors.lastname: Kadono
authors.forename: M
authors.initials: M
authors.lastname: Sakurai
authors.forename: H
authors.initials: H
authors.lastname: Moriyama
authors.forename: N
authors.initials: N
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology; D016963:Thermus thermophilus / Q000201:enzymology*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D013774:Tetranitromethane