l-Asparaginase from Erwinia carotovora

The enzymatic properties of Erwinia carofovora L-asparaginase have been investigated and the K, and V,,, values for a number of substrates determined.L-Asparaginase has a K,,,of ~10~~ and a V max of 0.9 pmole of ammonia released per min per unit of enzyme for L-asparagine.All other substrates have much higher K, values and less than 25% of the V,,, of L-asparagine.From a study of substrate analogues, it has been established that Erwinia L-asparaginase requires a carboxyl group on the substrate two or three carbon atoms from the carboxamide group which is hydrolyzed.An amino group alpha to the free carboxyl group and in the L-configuration increases the stability of the enzyme-substrate complex but is not essential for catalysis.Small peptides with carboxyl-terminal asparagine residues are deamidated but amino-terminal or internal asparagine residues and large peptides with carboxyl-terminal asparagine residues such as insulin or insulin A chain derivatives are not deamidated.Likewise, glutamine peptides are not deamidated.,&Aspartoalkylamides have been prepared and tested as substrates.Compounds with straight chain alkyl substituents were hydrolyzed, whereas compounds with branched alkyl groups were not hydrolyzed.In addition, @-aspartamidoglycopeptides were not hydrolyzed.Erwinia L-asparaginase catalyzed the exchange of IsO between water and aspartic acid and the hydroxylaminolysis of asparagine and aspartic acid by hydroxylamine.Since the pioneering work of Kidd (1) and of Broome (2), Gasparaginase has been shown to be a powerful antilymphosarcoma agent when used against an asparagine-requiring sarcoma.Although the enzyme has been found in a number of organisms, e.g.serum of guinea pig and related rodents (3)) chicken liver (4)) yeast and molds (5), plants (6), and a number of bacteria (7-9), not all of these enzymes are clinically active (10,11).The antitumor activity has been proposed to be a function of the circulation half-life of the enzyme in the host serum ( 12) and of the enzyme affinity for L-asparagine (13).Two of the enzymes from