Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15.
A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D004229:Dithiothreitol / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D006640:Histidine Decarboxylase / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011511:Proteus / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013053:Spectrophotometry
D006639:Histidine; D011732:Pyridoxal Phosphate; D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase; D004229:Dithiothreitol