Jakob C G, Lewinski K, LaCount M W, Roberts J, Lebioda L
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Biochemistry. 1997 Jan 28;36(4):923-31. doi: 10.1021/bi961979x.
Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D- and L-isomers of glutamine and asparagine. X-ray quality type-1 crystals of PGA have been obtained from 2.0 M ammonium sulfate. The space group is C222(1) with unit-cell dimensions a = 78.62, b = 135.80, and c = 137.88 A. The tetrameric molecule is located on a crystallographic 2-fold axis, and two subunits form the asymmetric portion of the unit cell. The structure was solved by the molecular replacement method and refined at 1.7 A resolution to an R = 19.9% with a good geometry of the model, G = 0.05. The resultant electron density maps enabled us to resolve individual constituent atoms of most residues and introduce minor revisions to the amino acid sequence. The catalytic loop, Thr20-Gly40, is in the closed conformation with excellent electron density in both subunits. A sulfate ion and an ammonium ion are bound in the substrate binding site and interect with the loop. This interaction appears to be responsible for the observed closed conformation. New arguments supporting Thr20 as the catalytic nucleophile in the asparaginase activity are proposed.
铜绿假单胞菌7A谷氨酰胺酶-天冬酰胺酶（PGA）催化谷氨酰胺和天冬酰胺的D型和L型异构体的水解反应。已从2.0 M硫酸铵中获得了X射线质量的PGA 1型晶体。空间群为C222(1)，晶胞参数a = 78.62、b = 135.80和c = 137.88 Å。四聚体分子位于晶体学2重轴上，两个亚基构成晶胞的不对称部分。该结构通过分子置换法解析，并在1.7 Å分辨率下精修至R = 19.9%，模型几何结构良好，G = 0.05。所得电子密度图使我们能够分辨大多数残基的单个组成原子，并对氨基酸序列进行微小修正。催化环，即Thr20-Gly40，在两个亚基中均处于封闭构象且具有良好的电子密度。一个硫酸根离子和一个铵离子结合在底物结合位点并与该环相互作用。这种相互作用似乎是观察到的封闭构象的原因。提出了支持Thr20作为天冬酰胺酶活性中催化亲核试剂的新论据。
