Oxidatively induced Cu for Mn exchange in protein phosphatase 1gamma: a new method for active site analysis.
Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.
Miyazaki
Atsushi
A
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
Sydnes
Magne O
MO
Isobe
Minoru
M
Ohinata
Hiroshi
H
Miyazu
Motoi
M
Takai
Akira
A
D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D054645:Protein Phosphatase 1 / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D017382:Reactive Oxygen Species; D008345:Manganese; D006639:Histidine; D003300:Copper; D054645:Protein Phosphatase 1