Stereochemical aspects of the nucleophilic attack in different classes of L-asparaginases

L-Asparaginases hydrolyze L-asparagine to L-aspartate with the release of ammonia. Currently, three completely unrelated structural classes of L-asparaginases are known, further subdivided into five types. In each class, the hydrolysis reaction is thought to proceed via a nucleophilic attack of an activated Thr or Ser residue on the carbonyl Csp² atom of the substrate amide group. With the possible exception of class 2 L-asparaginases, which function as N-terminal nucleophile (Ntn) hydrolases, the identity of the nucleophilic residue is, or at least has been historically, the subject of some controversy, and even in class 2 this issue may not be so entirely obvious. Structural chemistry has, however, excellent tools to figure out reaction mechanisms, based on the application of Bürgi's structure correlation method (SCM). Its principle allows one to predict the reaction trajectory if sufficient structural (crystallographic) examples of the reagents along the reaction path are known. With respect to the nucleophilic attack on a carbonyl group, the stereochemistry is governed by the nucleophile...electrophile distance and the Bürgi-Dunitz angle, later supplemented with the Flippin-Lodge angle. The latter angle is shown to be a poor parameter and is better replaced by the Herschlag dihedral between the planes of the attacking nucleophile and the attacked electrophile. In structural enzymology, applicability of the SCM principle requires the availability of structural examples of the enzyme in question in complex with the substrate or product of the catalytic reaction. In this work, we applied the SCM concept to the three classes of L-asparaginases, identifying in each case the most probable nucleophilic residue as Thr12 in EcAII (class 1), Thr179 in EcAIII (class 2) and Ser48 in ReAV (class 3). In addition, we applied the SCM analysis to the newly identified group of asparaginases without proper classification, called short-chain asparaginases, providing a basis for their proper affiliation in class 1. Finally, the SCM analysis shows that the chirality of the nucleophilic attack in class 2 asparaginases (pro-R) is opposite to that in all other asparaginases.