PALO VERDE (PARKINSONIA ACULEATA L) SEED AMINOACYLASE

1. 1. N-Acylamino acid amidohydrolase (EC 3.5.1.14), or aminoacylase, was isolated from the seeds of Palo Verde (Parkinsonia aculeata L.). The enzyme was localized within the cotyledons and embryo of the seed. An acetone powder preparation from the combined cotyledons and embryo furnished an active extract which was purified greater than 75-fold. Fractionation consisted of treatment with (NH4)2SO4, cold acetone, freeze-thaw (which removed a cold-labile, inhibitory protein), and chromatography on DEAE-Sephadex A-25. The specific activity was 2650 μmoles/h per mg protein nitrogen. 2. 2. Co2+ was shown to enhance activity and also to provide stability to the enzyme during the (NH4)2SO4 fractionation. 3. 3. N-Formyl-l-methionine was the best substrate and exhibited a vmax/Km = 2.9·105 at pH 7.2. The acetyl derivatives of l-methionine, l-valine, and l-leucine inhibited the enzyme at concentrations above 10 mM. The hydrolyses of N-acetyl-l-leucine and N-acetyl-l-valine exhibited non-Michaelis-Menten kinetics with an indication of two possible binding sites. 4. 4. The molecular weight was estimated by gel filtration to be 79 500. 5. 5. The enzyme was stable between pH 6.5 and 10.0, and up to a temperature of 50° for 10 min. The temperature optimum was 52°. 6. 6. The enzyme catalyzed the hydrolysis of P-nitrophenyl acetate and was inhibited by some classical sulfhydryl reagents. Mercaptoethanol enhanced enzyme activity. © 1969.