Argenine decarboxylase from Escherichia coli. I. Purification and specificity for substrates and coenzyme.

The inducible arginine decarboxylase of Escherichia coli, a pyridoxal-P enzyme, has been purified to a specific activity of 410 pmoles of COz per min per mg. It can be crystallized from ammonium sulfate solutions without increase in specific activity, sediments as a single species in the analytical ultracentrifuge, and shows only slight impurities on acrylamide gel electrophoresis. Its preferred substrate is L-arginine (Km, 0.65 mM, vmx, 500 pmoles per min per mg), but L-canavanine (K,, 1.2 mM) also is attacked at a rate 40y0 of that for L-arginine. Several other guanido and ureido derivatives are not attacked by the enzyme but do inhibit it competitively; the most effective of these inhibitors are cr-chloro-3-guanidovaleric acid (Ki, 0.026 mM) and cr-hydroxy-d-guanidovaleric acid (Ki, 0.19 mM). The pH optimum of arginine decarboxylase is 5.2 and the temperature optimum is 3740”. Its absorption spectrum has maxima at 280 and 420 mp and is not affected by variations in pH between 5.2 and 9.0. The formyl group of pyridoxal-P in arginine decarboxylase is bound to the t-amino group of a lysine residue. The holodecarboxylase can be resolved by dialysis against cysteine and reconstituted by addition of excess pyridoxal-P; it binds 10 moles of pyridoxal-P per mole (850,000 g). The apoenzyme is also reactivated by 2-norpyridoxal-P, w-methylpyridoxalP, and 6-methylpyridoxal-P, although its afhnity for these pyridoxal-P analogues and the affinity of the resulting analogue holoenzymes for arginine are reduced by these structural alterations. Pyridoxal, 5-deoxypyridoxal, 2-nor2-butylpyridoxal-P, and P-(2-methyl-3-hydroxy-4-formylpyridine-5)-propionic acid do not reactivate the apoenzyme but do inhibit its reactivation by pyridoxal-P when present in sufficient concentrations.