Structural characterization of Pseudomonas 7A glutaminase-asparaginase.

The amino acid sequence and a 2-A-resolution crystallographic structure of Pseudomonas 7A glutaminase-asparaginase (PGA) have been determined. PGA, which belongs to the family of tetrameric bacterial amidohydrolases, deamidates glutamine and asparagine. The amino acid sequence of PGA has a high degree of similarity to the sequences of other members of the family. PGA has the same fold as other bacterial amidohydrolases, with the exception of the position of a 20-residue loop that forms part of the active site. In the PGA structure presented here, the active site loop is observed clearly in only one monomer, in an open position, with a conformation different from that observed for other amidohydrolases. In the other three monomers the loop is disordered and cannot be traced. This phenomenon is probably a direct consequence of a very low occupancy of product(s) of the enzymatic reaction bound in the active sites of PGA in these crystals. The active sites are composed of a rigid part and the flexible loop. The rigid part consists of the residues directly involved in the catalytic reaction as well as residues that assist in orienting the substrate. Two residues that are important for activity residue on the flexible loop. We suggest that the flexible loops actively participate in the transport of substrate and product molecules through the amidohydrolase active sites and participate in orienting the substrate molecules properly in relation to the catalytic residues.

[1]  M. Jaskólski,et al.  Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M P Gallagher,et al.  Asparaginase as a drug for treatment of acute lymphoblastic leukaemia. , 1989, Essays in biochemistry.

[3]  K. Röhm,et al.  On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies. , 1989, Biochimica et biophysica acta.

[4]  M. L. Connolly Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[5]  A T Brünger,et al.  Slow-cooling protocols for crystallographic refinement by simulated annealing. , 1990, Acta crystallographica. Section A, Foundations of crystallography.

[6]  T. Copeland,et al.  Identification and characterization of SET, a nuclear phosphoprotein encoded by the translocation break point in acute undifferentiated leukemia. , 1994, The Journal of biological chemistry.

[7]  J. Holcenberg,et al.  Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes. , 1978, Biochemistry.

[8]  K. Röhm,et al.  A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis , 1991, FEBS letters.

[9]  L. H. Jensen,et al.  X-Ray Structure Determination: A Practical Guide , 1989 .

[10]  S. Shifrin,et al.  L-Asparaginase from Escherichia coli B. Physicochemical studies of the dissociation process. , 1971, The Journal of biological chemistry.

[11]  S. Abrahams,et al.  Normal probability plot analysis of error in measured and derived quantities and standard deviations , 1971 .

[12]  J. Howard,et al.  L-asparaginase from Erwinia carotovora. Substrate specificity and enzymatic properties. , 1972, Journal of Biological Chemistry.

[13]  Wolfgang Kabsch,et al.  Evaluation of Single-Crystal X-ray Diffraction Data from a Position-Sensitive Detector , 1988 .

[14]  A. Wlodawer,et al.  Characterization of crystals of L-glutaminase-asparaginase from Acinetobacter glutaminasificans and Pseudomonas 7A. , 1977, Journal of molecular biology.

[15]  D. Blow,et al.  Role of a Buried Acid Group in the Mechanism of Action of Chymotrypsin , 1969, Nature.

[16]  W. Dolowy,et al.  Isolation, crystallization, and properties of Achromobacteraceae glutaminase-asparaginase with antitumor activity. , 1972, The Journal of biological chemistry.

[17]  D. Miller,et al.  Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora. , 1972, The Biochemical journal.

[18]  M. Gribskov,et al.  A left‐handed crossover involved in amidohydrolase catalysis , 1993, FEBS letters.

[19]  A. Wlodawer,et al.  The molecular symmetry of glutaminase‐asparaginases: rotation function studies of the Pseudomonas 7A and Acinetobacter enzymes , 1983 .

[20]  F. Richards,et al.  INTERMOLECULAR CROSS LINKING OF A PROTEIN IN THE CRYSTALLINE STATE: CARBOXYPEPTIDASE-A. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[21]  N. Kitron,et al.  Stereospecific features of the conformative response of L-asparaginase. , 1972, Biochemistry.

[22]  B. Finzel Incorporation of fast Fourier transforms to speed restrained least‐squares refinement of protein structures , 1987 .

[23]  W. Mcgregor,et al.  Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase. , 1991, The Journal of general virology.

[24]  J. Roberts Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7Z. , 1976, The Journal of biological chemistry.