Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease a as revealed by crystallographic study of three Phe 120 mutants at 1.4 Å resolution

Eri Chatani, Rikimaru Hayashi, Hideaki Moriyama, Tatzuo Ueki

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Abstract

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants - F120A, F120G, and F120W - were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only ∼ 1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

Original languageEnglish (US)
Pages (from-to)72-81
Number of pages10
JournalProtein Science
Volume11
Issue number1
DOIs
StatePublished - Jan 10 2002

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Pancreatic Ribonuclease
Hot Temperature
Denaturation
Enzymes
Enthalpy
Hydrolysis
Catalytic Domain
Thermodynamic stability
Crystal structure
Molecules
Proteins

Keywords

  • Accessible surface area
  • Active site
  • Crystal structure
  • His119
  • Ribonuclease A
  • Thermal stability

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

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title = "Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease a as revealed by crystallographic study of three Phe 120 mutants at 1.4 {\AA} resolution",
abstract = "The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants - F120A, F120G, and F120W - were analyzed up to a 1.4 {\AA} resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be {"}inside out{"} compared with that of wild-type RNase A. Only ∼ 1 {\AA} change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.",
keywords = "Accessible surface area, Active site, Crystal structure, His119, Ribonuclease A, Thermal stability",
author = "Eri Chatani and Rikimaru Hayashi and Hideaki Moriyama and Tatzuo Ueki",
year = "2002",
month = "1",
day = "10",
doi = "10.1110/ps.ps.31102",
language = "English (US)",
volume = "11",
pages = "72--81",
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T1 - Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease a as revealed by crystallographic study of three Phe 120 mutants at 1.4 Å resolution

AU - Chatani, Eri

AU - Hayashi, Rikimaru

AU - Moriyama, Hideaki

AU - Ueki, Tatzuo

PY - 2002/1/10

Y1 - 2002/1/10

N2 - The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants - F120A, F120G, and F120W - were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only ∼ 1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

AB - The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants - F120A, F120G, and F120W - were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only ∼ 1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

KW - Accessible surface area

KW - Active site

KW - Crystal structure

KW - His119

KW - Ribonuclease A

KW - Thermal stability

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