Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Mechanism of inactivation by alpha-fluoromethylhistidine.
Mechanism-based inactivation of pyridoxal phosphate-dependent histidine decarboxylase by (S)-alpha-(fluoromethyl)histidine was studied. The molar ratio of inactivator to enzyme subunit required for complete inactivation increased from 1.63 at 10 degrees C to 3.00 at 37 degrees C. Two inactivation products were isolated by chromatographic fractionation of the reaction mixture and identified by NMR spectroscopy as 1-(4-imidazolyl)-3(5'-P-pyridoxylidene) acetone (I), the adduct formed between pyridoxal phosphate and inactivator, and 1-(4-imidazolyl) acetone (II), an intermediate compound formed during inactivation. Formation of these two products supports a previously proposed mechanism of inactivation (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009), with minor modifications. A precursor of I was linked covalently to the enzyme by NaBH4 reduction if the reaction was carried out immediately after inactivation, before development of the 403 nm peak of I. A mutant histidine decarboxylase (S322A) in which Ser-322 was changed to Ala was also inactivated by alpha-fluoromethylhistidine demonstrating that Ser-322 is not essential for inactivation even though it is close to the active site and is derivatized by borohydride reduction of the inactivated wild-type enzyme. Following inactivation, both the wild-type and the S322A mutant enzyme could be partially reactivated by prolonged dialysis against buffer.
Bhattacharjee
M K
MK
Department of Microbiology, University of Texas, Austin 78712.
Snell
E E
EE
D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives*; D006640:Histidine Decarboxylase / Q000037:antagonists & inhibitors* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008762:Methylhistidines / Q000494:pharmacology*; D015394:Molecular Structure; D009154:Mutation; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013053:Spectrophotometry
D008762:Methylhistidines; D011994:Recombinant Proteins; D006639:Histidine; D011732:Pyridoxal Phosphate; C028309:alpha-fluoromethylhistidine; D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase