The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing.
Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(III)-Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)-Fe(II) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-C-generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.
Klabunde
T
T
Institut für Anorganische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Stahl
B
B
Suerbaum
H
H
Hahner
S
S
Karas
M
M
Hillenkamp
F
F
Krebs
B
B
Witzel
H
H
D000135:Acid Phosphatase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001322:Autoanalysis; D001665:Binding Sites; D003488:Cyanogen Bromide; D007887:Fabaceae / Q000201:enzymology*; D006023:Glycoproteins / Q000737:chemistry*; D019811:Hydroxylamine; D006898:Hydroxylamines; D007501:Iron / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010946:Plants, Medicinal; D017421:Sequence Analysis / Q000379:methods*; D015032:Zinc / Q000737:chemistry*
D006023:Glycoproteins; D006898:Hydroxylamines; D010446:Peptide Fragments; D019811:Hydroxylamine; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D015032:Zinc; D003488:Cyanogen Bromide