The catalytic mechanism of L-asparaginase is a simple hydrolysis of amide bond of L-asparagine. The reaction can be assayed by measuring the release of ammonia in a simple Nessler test. L-asparaginase belongs to the aminohydrolase family and its mechanism was compared to serine protease family in which the activity depends on a set of amino acid residue namely the catalytic triad including three amino acids serine (Ser) as a nucleophilic residue, histidine (His) as a general base and Aspartic (Asp) as an acidic residue (Carter and Wells, 1988). Similarly, 3 residues of this protein including Thr89EcA (95ErA), Asp90EcA (96ErA) and Lys162EcA (168ErA) were initially suggested as the catalytic triad (Dodson and Wlodawer, 1998) in which Thr89EcA (95ErA) was proposed as the potential nucleophile basing on the crystallographic studies of EcA bound to L-asparaginase (Swain et al., 1993). This may be true because Thr89EcA (95ErA) which is more rigid would be the one making the first attack to nucleophile (Aghaiypour et al., 2001). Another key point, the action of aminohdrolysis acitivity was accessed by two sequential nucleophillic attacks of the paired active site threonine residues (Harms et al., 1991; Palm et al., 1996) Thr12EcA (15ErA) and Thr89EcA (95ErA). In fact, the remain thereonine residue Thr12EcA (15ErA) in flexbile loop is likely no acitivity but the recently study confirmed that mutation of Thr12EcA (15ErA) lead to remarkably decrease in the catalytic activity (Harms et al., 1991), and the binding of enzyme and aspartate was not affected. Therefore Thr12EcA (15ErA) is not the one binds to aspartate but acts as primary nucleophile of the enzyme (Palm et al., 1996). The most compelling evidence of the activation of primary attack of Thr12EcA (15ErA) is the involvement of Tyr25EcA (29ErA) as the enhancement for nucleophillic residue (Aung et al., 2000) by Glu283EcA (267ErA) which acts as the proton acceptor of Tyr25EcA (29ErA) (Ortlund et al., 2000). Thus, Thr89EcA (95ErA) acts as the second triad Thr89EcA (95ErA)-Asp90EcA (96ErA)-Lys162EcA (168ErA) and play an important role in the transitional state which responsible for the release of the product by cleave the scissile bond.
A proximal Lys162EcA (168ErA), which is stabilized by Asp90EcA (96ErA) acts as a base to enhance the nucleophilicity of the catalytic Thr89EcA (95ErA) residue in the acyl-enzyme intermediate. three catalytic triad residues all connected by the hydrogen bonds. In the beginning, the L-asparagine substrate binds to high nucleophilicity residue Thr12EcA, one proton is transferred from Thr89EcA (95ErA) to Asp90EcA (96ErA). The substrate forms a tetrahedral (the first) transition state with the enzyme, stabilization of the negative charge that develops on the Oxygen atom of the amide group during the stransition state which is achieved by an “oxyanion hole” resulting from the interaction with adjacent bond donor. One proton is transferred to amide group of substrate, which is released by the cleave of C-N bond, the enzyme is bound to remain part of substrate through acyl-linkage. Next, a water molecule bind to the enzyme through hydrogen bond in place of departed amide group. The water molecule transfers its proton to Asp90EcA (96ErA) and its hydroxyl group to remaining substrate fragment-the ester C atom of residue Asp. Again, a tetrahedral state (second) is formed, the residue Asp of L-asparagine is released, the acyl bond is cleaved, the proton is transfered back from Asp90EcA (96ErA) to Thr89EcA (95ErA) and the enzyme returns to its initial state
References
Aghaiypour, K., Wlodawer, A., and Lubkowski, J. (2001). Do bacterial L-asparaginases utilize a catalytic triad Thr-Tyr-Glu? Biochi, 1550, 117-128.
Carter, P., and Wells, J.A. (1988). Dissecting the catalytic triad of a serine protease. Nature. 332, 564-568.
Dodson, G., and Wlodawer, A. (1998). Catalytic triads and their relatives. Trends. Biochem. Sci. 23, 347-352.
Harms, E., Wehner, A., Aung, H.P., and Rohm, K.H. (1991). A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis. FEBS Lett. 285, 55-58.
Ortlund, E., Lacount, M.W., Lewinski, K., and Lebioda, L. (2000). Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu. Biochemistry. 39, 1199-1204.
Palm, G.J., Lubkowski, J., Derst, C., Schleper, S., Rohm, K.H., and Wlodawer, A. (1996). A covalently bound catalytic intermediate in Escherichia coli asparagianse: crystal structure of a Thr-89-Val mutant. FEBS Lett. 390, 211-216.
Swain, A.L., Jaskólski, M., Housset, D., Rao, J.K., and Wlodawer, A. (1993). Crystal strurcture of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc. Natl. Acad. Sci. USA. 90, 1474-1478
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