When grown with pantothenate as a sole source of carbon and nitrogen, Pseudowonas P-2 contains an inducible enzyme, pantothenate hydrolase, that hydrolyzes pantothenate to pantoic acid and p-alanine. By ammonium sulfate fractionation and gel filtration, the hydrolase was purified about 370-fold from disrupted cells of Ps. P-2. The enzyme funct.ons optimally at 28" and is rapidly inactivated at temperatures above 30". Its K M value for pantothenate at its optimum pH of 7.4 is 5 t m . No diffusible cofactors are required. The activity of the enzyme in crude cell-free extracts is easily sufficient to justify the view that its action represents the first step in metabolic degradation of pantothenate by this organism. T he isolation by enrichment culture of a soil organism, Pseudomonas P-2, that utilizes pantothenate as a sole source of carbon and nitrogen has been described previously (Goodhue and Snell, 1966a,b). &Alanine and pantoic acid were among the products found in the spent culture medium, and it was conc:uded that the first step in degradative metabolism of the vitamin was cleavage of the amide bond. This study Ihows that this cleavage results through simple hydrolysis by an inducible amidase, pantothenate hydrolase. The enzyme is unusual in its sensitivity to heat.
Experimental ProcedureSource of Enzyme. Ps. P-2 was cultured in 4.5-5.0 1. lots of pantothenate medium from 2% inoculum with aeration, as described earlier (Goodhue and Snell, 1966a,b). At the end of the exponential phase of growth (12-40 hr at 30") cultures were cooled to &2", and the cells harvested by centrifugation, washixi twice at 0" with distilled water, suspended in 6.7 mM potassium phosphate (pH 7.4), and held at 0" umil used. These cell suspensions, containing approximal ely 200 mg of cells (dry weight) per ml, were disrupted by treatment at 0" in 25-ml lots in the M. S. E. Ultrasonic Disintegrator at 18-20 kc (1.1-1.4 amp) for 20 min, then centrifuged for 10-15 min at 27,000-37,000 X g. The residue was resuspended in buffer and again treated in this same fashion. The procedure was repeated (up to six times) until no additional enzyme was extracted. The combined supernatant fluids (protein 8-20 mg/ml) were stored at -36" until used.Assay of Pantothenate Hydrolase. Hydrolase activity was determined routinely by measuring the p-alanine formed after 1 hr at 28" in 1.0 ml of reaction mixture containing, at pH 7.4, 1.8-18 pmoles of potassium pantothenate, 0.92 pmole of potassium phosphate, and 0.1 ml of an appropriately diluted enzyme preparation. The reaction was stopped by heating at 100" for 3 min. One unit of enzyme is the amount which forms 1 pmole of p-alaninelmin under these conditions. Determination of p-Alanine. Initially p-alanine was separated from reaction mixtures by descending chromatography on paper with pyridine-water (65 :35) as solvent. The ninhydrin-reactive zones were cut out and the color was extracted with methanol and estimated photometrically (Klett colorimeter, 565-630 mp filter) against similarly treated standards ...
