An essential lysine in the substrate-binding site of ornithine carbamoyltransferase.
Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.
Valentini
G
G
Dipartimento di Biochimica, Università di Pavia, Italy.
De Gregorio
A
A
Di Salvo
C
C
Grimm
R
R
Bellocco
E
E
Cuzzocrea
G
G
Iadarola
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002221:Carbamyl Phosphate / Q000494:pharmacology; D004289:Dolphins; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D001894:Borohydrides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D002221:Carbamyl Phosphate; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D009954:Ornithine Carbamoyltransferase; D014357:Trypsin; D008239:Lysine