Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue.
When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.
D000642:Ammonia-Lyases / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D005056:Euglena gracilis / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008239:Lysine / Q000032:analysis*; D010084:Oxidation-Reduction; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D014576:Uroporphyrinogen III Synthetase / Q000037:antagonists & inhibitors
D001894:Borohydrides; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D011163:Hydroxymethylbilane Synthase; D014576:Uroporphyrinogen III Synthetase; D000642:Ammonia-Lyases; D008239:Lysine