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 Citations (Scopus)

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 1 1994

Fingerprint

3-Isopropylmalate Dehydrogenase
Mutagenesis
Chemical modification
Site-Directed Mutagenesis
Enzymes
Catalytic Domain
Tetranitromethane
Thermus
Nitration
X ray analysis
Catalysis
X-Rays
Oxidoreductases
Substrates

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Cite this

Chemical modification and site-directed mutagenesis of tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8. / Miyazaki, Kentaro; Kadono, Shojiro; Sakurai, Masahiro; Moriyama, Hideaki; Tanaka, Nobuo; Oshima, Tairo.

In: Protein Engineering, Design and Selection, Vol. 7, No. 1, 01.01.1994, p. 99-102.

Research output: Contribution to journalArticle

Miyazaki, Kentaro ; Kadono, Shojiro ; Sakurai, Masahiro ; Moriyama, Hideaki ; Tanaka, Nobuo ; Oshima, Tairo. / Chemical modification and site-directed mutagenesis of tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8. In: Protein Engineering, Design and Selection. 1994 ; Vol. 7, No. 1. pp. 99-102.
@article{ae1f522e74c14a6f960fbeee2ef00757,
title = "Chemical modification and site-directed mutagenesis of tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8",
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 {\aa} respectively. The results suggest that Arg94 is responsible for the enzyme catalysis",
keywords = "3-isopropylmalate dehydrogenase, Chemical modification, Site-directed mutagenesis, Tetranitromethane, Tyrosine",
author = "Kentaro Miyazaki and Shojiro Kadono and Masahiro Sakurai and Hideaki Moriyama and Nobuo Tanaka and Tairo Oshima",
year = "1994",
month = "1",
day = "1",
doi = "10.1093/protein/7.1.99",
language = "English (US)",
volume = "7",
pages = "99--102",
journal = "Protein Engineering, Design and Selection",
issn = "1741-0126",
publisher = "Oxford University Press",
number = "1",

}

TY - JOUR

T1 - Chemical modification and site-directed mutagenesis of tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8

AU - Miyazaki, Kentaro

AU - Kadono, Shojiro

AU - Sakurai, Masahiro

AU - Moriyama, Hideaki

AU - Tanaka, Nobuo

AU - Oshima, Tairo

PY - 1994/1/1

Y1 - 1994/1/1

N2 - 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

AB - 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

KW - 3-isopropylmalate dehydrogenase

KW - Chemical modification

KW - Site-directed mutagenesis

KW - Tetranitromethane

KW - Tyrosine

UR - http://www.scopus.com/inward/record.url?scp=0028042206&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028042206&partnerID=8YFLogxK

U2 - 10.1093/protein/7.1.99

DO - 10.1093/protein/7.1.99

M3 - Article

C2 - 8140100

AN - SCOPUS:0028042206

VL - 7

SP - 99

EP - 102

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 1

ER -