Isolation, Crystallization, and Properties of Achromobacteraceae Glutaminase-Asparaginase with Antitumor Activity

Crystalline glutaminase-asparaginase with antitumor activity was prepared from an Achromobacteraceae soil isolate organism. This enzyme has l-glutaminase and l-asparaginase activity in a ratio of 1.2:1. The purification procedure provides an over-all yield of 40 to 60% from crude cell-free extract to homogeneous glutaminase-asparaginase and is adaptable to large scale isolation of the enzyme. Glutaminase-asparaginase is crystallizable from aqueous alcohol solutions in the absence of heavy metal cations. The highest yields of enzyme were obtained when cells were grown aerobically in a basal synthetic medium composed of l-glutamic acid, ammonium sulfate, trace minerals, and phosphate buffer. Glutaminase-asparaginase content remained relatively constant when the organism was at temperatures between 15 and 25°. Above 25° the enzyme content decreased with increasing temperature. The isoelectric point by isoelectric focusing of glutaminase-asparaginase on ampholytes is 8.43. The specific activity of homogeneous enzyme is 190 ± 20 i.u. per mg of protein and the E1%280 is 10.2. No carbohydrate or phospholipid was detected in the enzyme. No disulfide or sulfhydryl groups appear to be present on the enzyme. The Km values for l-glutamine and l-asparagine are 5.8 ± 1.5 x 10-6 and 4.8 ± 1.4 x 10-6 m, respectively. Glutaminase-asparaginase catalyzes the hydrolysis of the d isomers of glutamine and asparagine at about one-third the rate of the l isomers. The enzyme is not inhibited by ethyl-enediaminetetraacetate (0.1 mm), ammonia (10 mm), l-glutamate (30 mm), or l-aspartate (30 mm). 6-Diazo-5-oxo-l-norleucine which is not a substrate for the enzyme irreversibly inactivates glutaminase-asparaginase at very low concentrations. In contrast the l and d isomers of 5-diazo-4-oxonorvaline are attacked by the enzyme and are considerably poorer inhibitors.