Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue.
Diethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme.
D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism*; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004296:Dopa Decarboxylase / Q000378:metabolism*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D005561:Formates; D006639:Histidine; D004296:Dopa Decarboxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004047:Diethyl Pyrocarbonate