Single-or dual-mode switching of semisynthetic ribonuclease S′ with an iminodiacetic acid moiety in response to the copper(II) concentration

Itaru Hamachi, Yasuhiro Yamada, Tomoaki Matsugi, Seiji Shinkai

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Ribonuclease S′ bearing iminodiacetic acid as a metal-binding site was designed and semisynthesized by self-assembly of native S-protein with chemically modified S-peptide. Iminodiacetic acid-appended amino acid (Ida4) was synthesized and incorporated into the S-peptide sequence by solid-phase peptide synthesis based on Fmoc chemistry at a single site or double sites of the solvent-exposed side of the S-peptide. Circular dichroism (CD) spectroscopy of these S-peptides confirmed that the CuII ion induced an increase or decrease of a-helix conformation depending on the replacement position. S-Peptide/S-protein titration monitored by conventional enzymatic activity and UV or CD spectroscopy demonstrated that various S-peptides form a stable complex (Ida4-RNase S′) with S-protein, except when Met13 and Asp14 are replaced with Ida4. In CuII titration and thermal denaturation experiments with single-site replacement mutants, CuII binding occurred at 1:1 stoichiometry of Ida4/CuII with perturbation of the α-helix conformation. Both 2:1 and 2:2 stoichiometries were achieved by addition of CuII ions to double-site mutants, and were dependent on the CuII concentration. Most importantly, the A6/E9Ida4-RNase S′ mutant shows cooperative binding of CuII ion with two Ida4; holoenzyme stability is enhanced at 2:1 stoichiometry, but at 2:2 stoichiometry, two Ida4 sites independently bind two CuII ions, and the mutant is destabilized. Other double mutants showed simple destabilization of 3D structure upon CuII binding. The response of the enzymatic activity of these Ida4-RNase S′ to the concentration of CuII ion was evaluated by the hydrolysis of polyuridiric acid catalyzed by RNase S′ mutants. The CuII-induced activity change of single and double mutants agreed well with the structural response to CuII, that is, the activity of A6/E9Ida4-RNase S′ was enhanced upon cooperative CuII binding at 2:1 stoichiometry and then suppressed at the 2:2 ratio. The activity of all other mutants was simply suppressed by CuII ions. These results represent successful switching of A6/E9Ida4-RNase S′ activity in dual mode, that is, suppression (OFF) or enhancement (ON), depending on the environmental CuII concentration. Thus it has been established that rational design of a metal-binding site can confer the dual mode of response to a metal cation on the structure and activity of an enzyme.

Original languageEnglish
Pages (from-to)1503-1511
Number of pages9
JournalChemistry - A European Journal
Volume5
Issue number5
Publication statusPublished - Dec 1 1999

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Peptides
Copper
Stoichiometry
Ions
Acids
Protein S
Circular dichroism spectroscopy
Metals
Binding sites
Proteins
Titration
Conformations
Bearings (structural)
Binding Sites
Holoenzymes
Denaturation
iminodiacetic acid
ribonuclease S
Ribonucleases
Self assembly

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

Cite this

Single-or dual-mode switching of semisynthetic ribonuclease S′ with an iminodiacetic acid moiety in response to the copper(II) concentration. / Hamachi, Itaru; Yamada, Yasuhiro; Matsugi, Tomoaki; Shinkai, Seiji.

In: Chemistry - A European Journal, Vol. 5, No. 5, 01.12.1999, p. 1503-1511.

Research output: Contribution to journalArticle

Hamachi, Itaru ; Yamada, Yasuhiro ; Matsugi, Tomoaki ; Shinkai, Seiji. / Single-or dual-mode switching of semisynthetic ribonuclease S′ with an iminodiacetic acid moiety in response to the copper(II) concentration. In: Chemistry - A European Journal. 1999 ; Vol. 5, No. 5. pp. 1503-1511.
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abstract = "Ribonuclease S′ bearing iminodiacetic acid as a metal-binding site was designed and semisynthesized by self-assembly of native S-protein with chemically modified S-peptide. Iminodiacetic acid-appended amino acid (Ida4) was synthesized and incorporated into the S-peptide sequence by solid-phase peptide synthesis based on Fmoc chemistry at a single site or double sites of the solvent-exposed side of the S-peptide. Circular dichroism (CD) spectroscopy of these S-peptides confirmed that the CuII ion induced an increase or decrease of a-helix conformation depending on the replacement position. S-Peptide/S-protein titration monitored by conventional enzymatic activity and UV or CD spectroscopy demonstrated that various S-peptides form a stable complex (Ida4-RNase S′) with S-protein, except when Met13 and Asp14 are replaced with Ida4. In CuII titration and thermal denaturation experiments with single-site replacement mutants, CuII binding occurred at 1:1 stoichiometry of Ida4/CuII with perturbation of the α-helix conformation. Both 2:1 and 2:2 stoichiometries were achieved by addition of CuII ions to double-site mutants, and were dependent on the CuII concentration. Most importantly, the A6/E9Ida4-RNase S′ mutant shows cooperative binding of CuII ion with two Ida4; holoenzyme stability is enhanced at 2:1 stoichiometry, but at 2:2 stoichiometry, two Ida4 sites independently bind two CuII ions, and the mutant is destabilized. Other double mutants showed simple destabilization of 3D structure upon CuII binding. The response of the enzymatic activity of these Ida4-RNase S′ to the concentration of CuII ion was evaluated by the hydrolysis of polyuridiric acid catalyzed by RNase S′ mutants. The CuII-induced activity change of single and double mutants agreed well with the structural response to CuII, that is, the activity of A6/E9Ida4-RNase S′ was enhanced upon cooperative CuII binding at 2:1 stoichiometry and then suppressed at the 2:2 ratio. The activity of all other mutants was simply suppressed by CuII ions. These results represent successful switching of A6/E9Ida4-RNase S′ activity in dual mode, that is, suppression (OFF) or enhancement (ON), depending on the environmental CuII concentration. Thus it has been established that rational design of a metal-binding site can confer the dual mode of response to a metal cation on the structure and activity of an enzyme.",
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AU - Hamachi, Itaru

AU - Yamada, Yasuhiro

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N2 - Ribonuclease S′ bearing iminodiacetic acid as a metal-binding site was designed and semisynthesized by self-assembly of native S-protein with chemically modified S-peptide. Iminodiacetic acid-appended amino acid (Ida4) was synthesized and incorporated into the S-peptide sequence by solid-phase peptide synthesis based on Fmoc chemistry at a single site or double sites of the solvent-exposed side of the S-peptide. Circular dichroism (CD) spectroscopy of these S-peptides confirmed that the CuII ion induced an increase or decrease of a-helix conformation depending on the replacement position. S-Peptide/S-protein titration monitored by conventional enzymatic activity and UV or CD spectroscopy demonstrated that various S-peptides form a stable complex (Ida4-RNase S′) with S-protein, except when Met13 and Asp14 are replaced with Ida4. In CuII titration and thermal denaturation experiments with single-site replacement mutants, CuII binding occurred at 1:1 stoichiometry of Ida4/CuII with perturbation of the α-helix conformation. Both 2:1 and 2:2 stoichiometries were achieved by addition of CuII ions to double-site mutants, and were dependent on the CuII concentration. Most importantly, the A6/E9Ida4-RNase S′ mutant shows cooperative binding of CuII ion with two Ida4; holoenzyme stability is enhanced at 2:1 stoichiometry, but at 2:2 stoichiometry, two Ida4 sites independently bind two CuII ions, and the mutant is destabilized. Other double mutants showed simple destabilization of 3D structure upon CuII binding. The response of the enzymatic activity of these Ida4-RNase S′ to the concentration of CuII ion was evaluated by the hydrolysis of polyuridiric acid catalyzed by RNase S′ mutants. The CuII-induced activity change of single and double mutants agreed well with the structural response to CuII, that is, the activity of A6/E9Ida4-RNase S′ was enhanced upon cooperative CuII binding at 2:1 stoichiometry and then suppressed at the 2:2 ratio. The activity of all other mutants was simply suppressed by CuII ions. These results represent successful switching of A6/E9Ida4-RNase S′ activity in dual mode, that is, suppression (OFF) or enhancement (ON), depending on the environmental CuII concentration. Thus it has been established that rational design of a metal-binding site can confer the dual mode of response to a metal cation on the structure and activity of an enzyme.

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