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^,

Abstract

Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism.

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.…”

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

“…Previous studies used intein-based chemistry to add electrophilic moieties such as vinyl methyl ester to the C terminus of ubiquitin to covalently trap nucleophilic cysteines in deubiquitinases. Because Dop has glutamine deamidase activity, we modified Pup with DON, a glutamine mimic produced by Streptomyces (21) that has been used to identify nucleophilic residues in glutamine-hydrolyzing enzymes (22,23). DON did not inhibit Dop-dependent amidohydrolysis in vitro (supplemental Fig.…”

Asparaginase – An Enzyme for Acrylamide Reduction in Food Products

“…Crystallographic analyses of microbial asparaginases and glutaminase-asparaginases have shown that they share the same basic structure and catalytic mechanism. 60 Glutamine Some microbes produce a periplasmic or extracellular asparaginase or glutaminaseasparaginase in addition to the intracellular asparaginase that participates in basic metabolism. [62][63][64][65] In the few cases where both enzymes have been studied, the two forms of asparaginase are similar in primary amino acid sequence and protein structure, but differ in other characteristics like pH and temperature activity profiles and, most notably, in substrate affinity.…”