Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide.
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.
Dominici
P
P
Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy.
Simmaco
M
M
Tancini
B
B
Barra
D
D
Borri Voltattorni
C
C
D000595:Amino Acid Sequence; D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D002851:Chromatography, High Pressure Liquid; D007460:Iodoacetamide / Q000378:metabolism / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013552:Swine; D014357:Trypsin
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; C067116:isoleucyl-histidyl-leucyl-valyl-prolyl-carboxyamidomethylcysteinyl-arginine; D014357:Trypsin; D007460:Iodoacetamide