Structural Basis for the Activity and Substrate Specificity of Erwinia chrysanthemi l-Asparaginase^,

Abstract

Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.

One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora

“…ErA and ECAR-LANS have similar V max values for the main physiological substrate, L-Asn (Table 2), which is not surprising in view of the high level of homology (78 %) between these enzymes [16]. The ECAR-LANS region between Thr 12 and Gly 19 corresponds to the highly conserved domain of the active-site flexible loop of bacterial asparaginases (Figure 3), including Thr 15 , which serves as an attacking nucleophile [4]. The side-chain group of a substrate need not necessarily be an amino group for effective catalysis.…”

“…The side-chain group of a substrate need not necessarily be an amino group for effective catalysis. The replacement of γ -NH 4 + of L-Asn with a hydroxylamide group (L-aspartyl-β-hydroxamate) decreased V max of ECAR-LANS and EcA2 only 3-fold. Yet the simultaneous increase of K m results in a 10-fold decrease of the specificity constants (k cat /K m ) of these enzymes (Table 2).…”

“…The disadvantage of this assay is that, because of the short lag period before the reaction reaches the steady state and the high K m value of glutamate dehydrogenase for ammonia, large quantities of this enzyme are required. In our study we used direct continuous monitoring of the absorbance changes occurring in NH 4 + release from the substrate. This method is easy, sensitive and readily reproducible.…”

“…The amount of NH 4 + was determined by the Nessler method [17]. L-Asparaginase activity was expressed in generally accepted international units (i.u.).…”

“…The active-site residues have been identified in the crystallographic studies of asparaginase from Eschericha coli [3]. The high-resolution structure of 0.17 nm (1.7 Å ) for L-asparaginase from Erwinia chrysanthemi elucidated the residues presumably responsible for different specificities towards two physiological substrates, L-Asn and L-Gln [4].…”