Asparaginase

I.U.B.: 3.5.1.1
L-Asparaginase amidohydrolase

L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia.

The enzyme is present in many animal tissues, bacteria and plants and in the serum of certain rodents but not of man.

Increasing interest in this enzyme stemmed from its identification by Broome (1961) as the antilymphoma factor of Kidd (1953). Following the finding that the E. coli enzyme had anti-tumor activity (Mashburn and Wriston 1963) work proceeded in several laboratories and clinics leading to clinical trials. (Clarkson et al. 1970; Oettgen et al. 1970; Boyse et al. 1967; Hill et al. 1967; Old et al. 1967; Roberts et al. 1966), particularly in acute lymphoblastic leukemia where it continues to be of benefit when used in sequential chemotherapy (Lay et al. 1975), (Bodey et al. 1974). See also Perez-Bercoff et al. (1973), Fidler et al. (1971) and Whitecar et al. (1970).

Friedman (1971) and Chakrabarty and Friedman (1970) studied suppression of the immune response of injected enzyme. See also Ryan and Curtis (1973), Prager and Derr (1970) and Berenbaum et al. (1970).

The enzyme is not without antigenicity (King et al. 1974) and toxicity (Rutter 1975) (Schrek et al. 1967).

Studies employing immobilized enzyme in extracorporeal devices have been reported: (Cooney et al. 1975; Inada et al. 1975; Chang 1971 and 1972-73; Mori et al. 1972 and 1973; Allison et al. 1972).

More recently, therapeutic applications of polyethylene glycol-asparaginase conjugates have been described. (MacEwen et al. 1987; Sur et al. 1987; Yoshimoto et al. 1986; and Abuchowski et al. 1984).

Asparaginase has been isolated from a number of sources: Proteus vulgaris (Lee and Yang 1973; Tosa et al. 1973 and 1972). Erwinia carotovora (Shifrin et al. 1973; Cammack et al. 1972). Acinetobacter ( Jones et al. 1973; Holcenberg et al. 1972). Serratia marcescens (Whelan and Wriston 1974); Mycobacterium bovis (Soru et al. 1972). Streptomyces griseus (DeJong 1972); Achromobacteraceae ( Roberts et al. 1972); guinea pig liver ( Mathews and Brown 1974); Pisum sativum (Siechiechowicz and Ireland 1989). Not all enzyme species possess anti-tumor properties which seem to be related to the affinity of enzyme for its substrate (Schwartz et al. 1966) and to factors affecting the clearance rate from the system (Broome 1965). Of the two asparaginases from E. coli (EC-1 and EC-2) only one (EC-2), possesses antilymphoma activity (Campbell et al. 1967). The Erwinia enzyme also has this property (King et al. 1974; Han and Ohnuma 1972).

The most extensively studied asparaginase is the EC-2 from E. coli which differs from EC-1 by its broad pH activity profile and its higher substrate affinity. EC-1 is usually eliminated during purification.

Characteristics of Asparaginase from E. coli:

Molecular weight: 141,000 (Maita et al. 1974), (Shifrin et al. 1974).

Composition: The enzyme is tetrameric (Marlborough et al. 1975; Shifrin and Parrott 1974; Greenquist and Wriston 1972; Epp et al. 1971; Frank et al. 1970). An amino acid sequence has been reported by Maita et al. (1974). See also Gumprecht and Wriston (1973), Wriston (1971), et al. (1971), Ho et al. (1970) and Kirschbaum et al. (1969). Studies on the active center have been reported by Shifrin et al. (1974), Makino and Inada (1973), and Coleman and Handschumacher (1973). See also Shifrin and Solis (1972), Makino et al (1972), Citri and Zyk (1972), Citri et al. (1972) Nishimura (1971), Cooney and Davis (1970), Jackson and Handschumacher (1970), and Ryoyama (1972).

Extinction coefficient: = 7.1 (Ho et al. 1971).

Constants: K_m = 1.25 X 10^-5 (Wriston 1971).

Isoelectric point: pH 4.9 (Wriston 1970).

Specificity: The purified enzyme shows 2-4% activity on glutamine and 5% on D-asparagine, Herrman et al. (1974) report on its action on derivatives and analogues of L-asparagine.

Inhibitors: 5-diazo-4-oxo-L-norvaline (DONV) (Jackson and Handschumacher 1970). This compound is also an alternate substrate for the enzyme.

Stability: Highly purified asparaginase is reported to be stabilized by addition of buffer salts or extra protein.

Two reviews by Wriston (1970 and 1971) summarize work on both the E. coli enzymes and the mammalian forms as well as those from other microbial sources.