Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine.
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.
Dipartimento di Medicina Sperimentale, Università di Perugia, Italy.
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Dominici
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Maras
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Mei
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Borri Voltattorni
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003545:Cysteine / Q000737:chemistry; D004296:Dopa Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000737:chemistry; D013552:Swine; D014357:Trypsin
D000345:Affinity Labels; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D011733:Pyridoxamine; C017087:bromoacetylpyridoxamine phosphate; D002918:Chymotrypsin; D014357:Trypsin; D004296:Dopa Decarboxylase; D003545:Cysteine