Metal-catalyzed oxidation of histidine in human growth hormone. Mechanism, isotope effects, and inhibition by a mild denaturing alcohol.
Metal-catalyzed oxidation of proteins represents an important pathway of post-translational modification. We utilized human growth hormone (hGH), a protein with a well defined metal-binding site, to study the detailed mechanism of metal-catalyzed oxidation by ascorbate/Cu(II)/O2. Particularly His18 and His21 within the metal-binding site were oxidized, predominantly to 2-oxo-His with the incorporated oxygen originating from molecular oxygen, based on amino acid analysis, tryptic mapping, mass spectrometry, isotopic labeling, and 1H NMR. The anaerobic reduction of a hGH/Cu(II) mixture by ascorbate generated a hGH-Cu(I) complex with NMR spectral features different from those of native hGH and hGH/Cu(II). The anaerobic reaction of this hGH-Cu(I) complex with hydrogen peroxide resulted in the oxidation of His18 and His21, suggesting that a fraction of Cu(I) was bound at the metal-binding site of hGH. Site-specific oxidation of hGH required an intact metal-binding site and could largely (about 80%) be inhibited by the presence of >/=28% (v/v) 1-propanol which appears (i) to perturb the metal-binding site and (ii) to interact with a reactive oxygen species formed at the perturbed metal-binding site. The inhibition by 1-propanol-d7 (CD3CD2CD2OH) was significantly lower than that by 1-propanol-h7 with [residual hGH]1-propanol-h7/[residual hGH]1-propanol-d7 = 1.95 at 30% (v/v) 1-propanol, reflecting a kinetic isotope effect close to that for the reaction of a hydroxyl radical with Calpha-H/D bonds of methanol, suggesting the involvement of a hydroxyl radical-like species in the oxidation of His.
Zhao
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F
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA.
Ghezzo-Schöneich
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E
Aced
G I
GI
Hong
J
J
Milby
T
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Schöneich
C
C
D000433:1-Propanol / Q000378:metabolism*; D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D015032:Zinc / Q000378:metabolism*
D019382:Human Growth Hormone; D006639:Histidine; D003300:Copper; D000433:1-Propanol; D006861:Hydrogen Peroxide; D015032:Zinc; D001205:Ascorbic Acid; C030973:cupric oxide