A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Abstract

Escherichia coli asparaginase |I catalyzes the hydrol-) sis of L-asparagine to L-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.:£ey words'." Asparaginase II; Acyl-enzyme intermediate; !'hreonine amidohydrolase; Enzymatic mechanism !. IntroductionAsparaginases catalyze the hydrolysis of L-asparagine to L~spartate and ammonia. The enzymes isolated from Escheri-'hia coli (EcA) and Erwinia chrysanthemi (ERA) have been ltilized as anti-leukemia drugs for many years [1]. Treatment vith asparaginases is often accompanied by severe side effects, vhich are partially attributed to the glutaminase activity of hese enzymes [2]. In order to understand the enzyme specifi-:ity and to ultimately eliminate the glutaminase activity, the nechanism of action must be elucidated. Several members of t larger family of homologous L-asparaginases have thus been horoughly investigated over many years.Results of kinetic measurements [3,4] indicated that the en-,ymatic reaction proceeds via a covalent intermediate, probtbly a ~-aspartyl enzyme (Fig. 1). This mechanism was con-,irmed by NMR studies with aspartate through the oxygen ~xchange reaction with bulk solvent at the side chain carboxdate [5]. These experiments showed that at pH < 5 L-aspartate ~ith a protonated side chain binds to the enzyme as tightly as ~-asparagine and it can also form an acyl-enzyme intermediate, subsequently hydrolyzed to L-aspartate. Thus, at low pH 3oth L-aspartate and L-asparagine can function as substrates. l'he nature and identity of the primary nucleophile of the mzyme participating in the formation of the acyl-enzyme in-*Corresponding author. Fax: (1) . Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx. 4x330 residues. The tetramer is more accurately described as a dimer of dimers. Each of two identical active sites in each dimer is formed by both monomers. In the structure the active sites have been observed with and without aspartate as ligand. Part of the active site is covered by a flexible loop that contains the two important residues Thr-12 and Tyr-25. The hydroxyl groups of Thr-12 and Thr-89 are closest to the side chain carboxylate of an aspartate bound in the active site. These residues are the most likely candidates for the primary nucleophile. Bacterial L-asparaginases were the first threonine amidohydrolases described in the literature. Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong t...

Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties

“…This new position brings Tyr 308Ј in close proximity to Thr 19 . The homologous threonine in the EcII L-asparaginase has previously been implicated in the reaction mechanism, forming a covalent intermediate with the substrate Asn (36). The potential role of this tyrosine in the hydrolysis reaction is discussed below.…”

Gln-tRNAGln Formation from Glu-tRNAGln Requires Cooperation of an Asparaginase and a Glu-tRNAGln Kinase

“…Instead, a threonine residue located in a flexible loop near the N terminus is used as the primary nucleophile by 2 H. D. Becker, personal communication. asparaginases (17,18), and a well conserved Thr-Asp-Lys triad is found in the substrate-binding pocket (14,15). Sequence analysis indicated that these active site residues are also conserved in GatD, and Thr-101, Thr-177, Asp-178, and Lys-254 in M. thermautotrophicus GatD may be important for asparagine/ glutamine recognition (Fig.…”

Overview on L-Asparaginase

“…[6] Certain studies showed slight variation compared to this observation. Studies on Streptomyces albidoflavus showed that optimum enzyme production occurred at a pH of 7.5 [129] while enzyme productivity by Nocardia levis was maximum at a pH 7.0. [75] The optimum pH for enzyme production in…”