메뉴 건너뛰기
Journal of the American Chemical Society
Volumn 131, Issue 31, 2009, Pages 11155-11160
How a single-point mutation in horseradish peroxidase markedly enhances enantioselectivity
Author keywords

[No Author keywords available]
Indexed keywords

ELECTROSTATIC REPULSION; ENANTIOSELECTIVE; ENZYMATIC OXIDATIONS; ENZYME-SUBSTRATE COMPLEXES; HORSERADISH PEROXIDASE; KINETIC ANALYSIS; SINGLE-POINT MUTATION; TRANSITION STATE ENERGIES; VARIOUS SUBSTRATES; WILD TYPES;
EID: 68249144231     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja903482u     Document Type: Article
Times cited : (7)
References (46)
  • 6
  • 27
    • 68249136228 scopus 로고    scopus 로고
    • Oxidation of fluorescent phenolic substrates by surface-bound HRP produces phenolic radicals that are captured by the cell surface, thereby fluorescently labeling the yeast cells. Fluorescence intensity of these cells, measured by flow cytometry, is directly proportional to the catalytic activity of the enzyme. An epitope tag fused to HRP allows quantitative determination of the enzyme concentration using fluorescently labeled antibodies
    • Oxidation of fluorescent phenolic substrates by surface-bound HRP produces phenolic radicals that are captured by the cell surface, thereby fluorescently labeling the yeast cells. Fluorescence intensity of these cells, measured by flow cytometry, is directly proportional to the catalytic activity of the enzyme. An epitope tag fused to HRP allows quantitative determination of the enzyme concentration using fluorescently labeled antibodies.
  • 28
    • 68249143832 scopus 로고    scopus 로고
    • Defined as the ratio of the initial rate of the enzymatic oxidation of the (S)-enantiomer divided by that of the (R)-enantiomer.
    • Defined as the ratio of the initial rate of the enzymatic oxidation of the (S)-enantiomer divided by that of the (R)-enantiomer.
  • 29
    • 68249156088 scopus 로고    scopus 로고
    • This supposition is supported by computational docking of these HRP variants with 1 which indicates that the substitution of Arg178 with bulky aromatic residues makes the active site inaccessible to 1 data not shown, We are unable to measure catalytic activity of these variants using smaller phenols, as our methodology does not allow characterization of the surface-bound enzyme using non-fluorescent substrates
    • This supposition is supported by computational docking of these HRP variants with 1 which indicates that the substitution of Arg178 with bulky aromatic residues makes the active site inaccessible to 1 (data not shown). We are unable to measure catalytic activity of these variants using smaller phenols, as our methodology does not allow characterization of the surface-bound enzyme using non-fluorescent substrates.
  • 30
    • 0030734513 scopus 로고    scopus 로고
    • The increased enantioselectivity of HRP variants with amino acids substitutions that remove the positive charge at position 178 may be explained by the presence of a putative cation-π interaction between Arg178 (or Lys178) and Phe179. Elimination of this interaction shifts the position of Phe179, a residue shown to be critical in substrate binding: Veitch, N. C, Gao, Y, Smith, A. T, White, C. G. Biochemistry 1997, 36, 14751sthereby sterically hindering binding of 1 and, in turn, influencing the enantioselectivity of these variants
    • The increased enantioselectivity of HRP variants with amino acids substitutions that remove the positive charge at position 178 may be explained by the presence of a putative cation-π interaction between Arg178 (or Lys178) and Phe179. Elimination of this interaction shifts the position of Phe179 - a residue shown to be critical in substrate binding: Veitch, N. C.; Gao, Y.; Smith, A. T.; White, C. G. Biochemistry 1997, 36, 14751sthereby sterically hindering binding of 1 and, in turn, influencing the enantioselectivity of these variants.
  • 31
    • 68249150149 scopus 로고    scopus 로고
    • It is also possible, however, that the sulfonates are not involved because they form intramolecular salt bridges with the neighboring protonated amino groups
    • It is also possible, however, that the sulfonates are not involved because they form intramolecular salt bridges with the neighboring protonated amino groups.
  • 32
    • 0029016182 scopus 로고    scopus 로고
    • Altering the strength of electrostatic interactions by varying salt concentration in solution is commonly used in enzyme studies, e.g. Honig, B, Nicholls, A. Science 1995, 268, 1144
    • Altering the strength of electrostatic interactions by varying salt concentration in solution is commonly used in enzyme studies, e.g. Honig, B.; Nicholls, A. Science 1995, 268, 1144.
  • 33
    • 34247853302 scopus 로고    scopus 로고
    • In contrast to such conventional electrostatic interactions as salt bridges, cation-π interactions have been shown to be essentially insensitive to salt concentration in solution: Berry, B. W.; Elvekrog, M. M.; Tommos, C. J. Am. Chem. Soc. 2007, 129, 5308.
    • In contrast to such conventional electrostatic interactions as salt bridges, cation-π interactions have been shown to be essentially insensitive to salt concentration in solution: Berry, B. W.; Elvekrog, M. M.; Tommos, C. J. Am. Chem. Soc. 2007, 129, 5308.
  • 34
    • 68249151615 scopus 로고    scopus 로고
    • It should be noted that the enantioselectivity of the Arg178Glu enzyme toward 3 differs from that of the wild-type enzyme (2.4 ( 0.1 Vs 1.1 ( 0.1, respectively) caused by a surprising doubling in the oxidation rate of the (S)-enantiomer (Table 1).
    • It should be noted that the enantioselectivity of the Arg178Glu enzyme toward 3 differs from that of the wild-type enzyme (2.4 ( 0.1 Vs 1.1 ( 0.1, respectively) caused by a surprising doubling in the oxidation rate of the (S)-enantiomer (Table 1).
  • 36
    • 68249148639 scopus 로고    scopus 로고
    • m regime is at play.
    • m regime is at play.

* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.