Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases

Mario      Sanches; Sandra      Krauchenco; Igor      Polikarpov

doi:10.2174/2212796810701010075

Abstract

Asparaginases and glutaminases are enzymes that catalyze the hydrolysis of asparagine or glutamine to the correspondent acid and ammonia. Based on their biochemical properties and sequence homology, this group of proteins, common to various organisms, can be divided into three families: bacterial asparaginases, plant asparaginases and enzymes similar to Rhizobium etli asparaginase. Bacterial L-asparaginases can be further subdivided into two types: type I, which are expressed constitutively and display enzymatic activity towards both L-asparagine and L-glutamine, and type II, induced by anaerobic conditions, which have high specific activity towards L-asparagine. Type II L-asparaginases (e.g. E. coli L-asparaginase) have been used in the treatment of acute lymphoblastic leukemia for many years, but their medical applications are limited by severe side effects and by the development of resistant tumors in a fraction of the patients. In this paper we review available structural and biochemical information on bacterial L-asparaginases, and focus on a detailed mechanistic description of their reaction mechanism, including the structural basis for the preference of these enzymes for threonine residues as the primary nucleophiles. The L-asparaginase enzymatic mechanism involves two catalytic triads operating at distinct steps of the reaction pathway. The first triad, Thr12-Tyr25-Glu283 (E. coli asparaginase numbering), acts during the acylation step starting with a nucleophilic attack of the primary nucleophile (Thr12) on the substrate, which results in an intermediate covalently bound to the enzyme. The second triad, Thr89-Lys162-Asp90, acts by activating a water molecule, which releases the product through a second nucleophilic attack. A detailed comprehension of the enzymatic mechanism of these bacterial enzymes in structural terms might open the way to design modified Lasparaginases with improved biomedical and biotechnological properties.

Keywords: Asparaginase, glutaminase, enzymatic mechanism, substrate recognition, leukemia treatment

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