CATALYTIC CONFORMATION OF PSEUDOMONAS 7A GLUTAMINASE-ASPARAGINASE (PGA): CRYSTAL STRUCTURE OF THE PGA-SO₄^2--NH₄⁺ COMPLEX AT 1.7 Å RESOLUTION. C. Jakob¹, M. LaCount², K. Lewinski³, J. Roberts⁴, L. Lebioda^{2 1}Chemistry, Davidson College, Davidson, NC 28036, USA, ²Chemistry and Biochemistry, Univ. of South Carolina, Columbia, SC 29208,USA, ³Chemistry, Jagiellonian Univ., Cracow, Poland, ⁴Pharmacy, Univ. of South Carolina, SC 29208, USA

Pseudomonas 7A Glutaminase-Asparaginase (PGA) catalyzes the hydrolysis of D- and L- isomers of glutamine and asparagine. Type-1 crystals of PGA have been obtained from high salt concentrations. The space group is C222₁ with unit-cell dimensions a = 78.62, b = 135.80, and c = 137.88Å. X-ray diffraction data set was collected on an R-Axis IV area detector and is 86% complete to 1.71Å with 68,971 reflections and R_merge = 7.5%. The molecular replacement method with Escherichia coli L-Asparaginase model was employed to solve the crystallographic structure of PGA at 1.7Å resolution. The resultant high resolution electron density maps enabled us to introduce minor revisions to the amino acid sequence. Each subunit of PGA has an active site consisting of a relatively rigid region and a flexible loop. The catalytic triad, which is analogous to those of serine proteases, has been previously assigned to residues Thr100, Asp101, and Lys173 and are located in the relatively rigid portion of the active site. Earlier published structures of PGA report the flexible loop (residues Thr20-Gly40) in either an open conformation or as a partially disordered region. In the PGA structure reported here, the active site flexible loop is in the closed conformation in both subunits and excellent electron density is observed. This conformation is induced by the presence of sulfate and ammonium ions in the active site. This suggests that the process of loop closure is driven by electrostatic interactions.