Site-directed mutagenesis of histidine 238 in mouse adenosine deaminase

Substitution of histidine 238 does not impede hydroxylate formation

Vera Sideraki, David K. Wilson, Linda C. Kurz, Florante A. Quiocho, Frederick B. Rudolph

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

His 238, a conserved amino acid located in hydrogen-bonding distance from C-6 of the substrate in the active site of murine adenosine deaminase (mADA) and postulated to play an important role in catalysis, was altered into an alanine, a glutamate, and an arginine using site-directed mutagenesis. The Ala and Glu substitutions did not result in changes of the secondary or tertiary structure, while the Arg mutation caused local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans. Neither the Glu or Arg mutations affected substrate binding affinity. By contrast, the Ala mutation enhanced substrate and inhibitor binding by 20-fold. The most inactive of the mutants. Glu 238, had a k(cat)/K(m) 4 x 10-6 lower than the wild-type value, suggesting that a positive charge on His 238 is important for proper catalytic function. The Ala 238 mutant was the most active ADA, with a k(cat)/K(m) 2 x 10-3 lower than the wild-type value. NMR spectroscopy and crystallography revealed that this mutant is able to catalyze hydration of purine riboside, a ground state analog of the reaction. These results collectively show that His 238 is not required for formation of the hydroxylate used in the deamination and may instead have an important electrostatic role.

Original languageEnglish (US)
Pages (from-to)15019-15028
Number of pages10
JournalBiochemistry
Volume35
Issue number47
DOIs
StatePublished - Nov 26 1996
Externally publishedYes

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Mutagenesis
Site-Directed Mutagenesis
Histidine
Substitution reactions
arginine glutamate
Mutation
Substrates
Deamination
Crystallography
Adenosine Deaminase
Hydrogen Bonding
Static Electricity
Catalysis
Tryptophan
Hydration
Alanine
Ground state
Nuclear magnetic resonance spectroscopy
Electrostatics
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of histidine 238 in mouse adenosine deaminase : Substitution of histidine 238 does not impede hydroxylate formation. / Sideraki, Vera; Wilson, David K.; Kurz, Linda C.; Quiocho, Florante A.; Rudolph, Frederick B.

In: Biochemistry, Vol. 35, No. 47, 26.11.1996, p. 15019-15028.

Research output: Contribution to journalArticle

Sideraki, Vera ; Wilson, David K. ; Kurz, Linda C. ; Quiocho, Florante A. ; Rudolph, Frederick B. / Site-directed mutagenesis of histidine 238 in mouse adenosine deaminase : Substitution of histidine 238 does not impede hydroxylate formation. In: Biochemistry. 1996 ; Vol. 35, No. 47. pp. 15019-15028.
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abstract = "His 238, a conserved amino acid located in hydrogen-bonding distance from C-6 of the substrate in the active site of murine adenosine deaminase (mADA) and postulated to play an important role in catalysis, was altered into an alanine, a glutamate, and an arginine using site-directed mutagenesis. The Ala and Glu substitutions did not result in changes of the secondary or tertiary structure, while the Arg mutation caused local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans. Neither the Glu or Arg mutations affected substrate binding affinity. By contrast, the Ala mutation enhanced substrate and inhibitor binding by 20-fold. The most inactive of the mutants. Glu 238, had a k(cat)/K(m) 4 x 10-6 lower than the wild-type value, suggesting that a positive charge on His 238 is important for proper catalytic function. The Ala 238 mutant was the most active ADA, with a k(cat)/K(m) 2 x 10-3 lower than the wild-type value. NMR spectroscopy and crystallography revealed that this mutant is able to catalyze hydration of purine riboside, a ground state analog of the reaction. These results collectively show that His 238 is not required for formation of the hydroxylate used in the deamination and may instead have an important electrostatic role.",
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