Chemical modification and site-directed mutagenesis of tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8

Kentaro Miyazaki, Shojiro Kadono, Masahiro Sakurai, Hideaki Moriyama, Nobuo Tanaka, Tairo Oshima

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

3-Isopropylmalate dehydrogenase from an extreme thermo-phile, Thermus thermophUus HB8, was chemically modified with tetranitromethane which nitrated 1.5-2.0 Tyr residues per subunit. The nitration was biphask 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 -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 å respectively. The results suggest that Arg94 is responsible for the enzyme catalysis

Original languageEnglish (US)
Pages (from-to)99-102
Number of pages4
JournalProtein Engineering, Design and Selection
Volume7
Issue number1
DOIs
StatePublished - Jan 1994

Keywords

  • 3-isopropylmalate dehydrogenase
  • Chemical modification
  • Site-directed mutagenesis
  • Tetranitromethane
  • Tyrosine

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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