Crystallographic and biochemical studies of Pseudomonas 7A glutaminase-asparaginase, human prostatic acid phosphatase, human complement C8gamma and chicken annexin V

Chapter 1. Pseudomonas 7A Glutaminase-Asparaginase (PGA) catalyzes the hydrolysis of D- and L- isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogs of glutamine (6-diazo-5-oxo-L-norleucine - DON) and asparagine (5-diazo-4-oxo-L-norvaline - DONV). 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.; Chapter 2. C8γ is a 22 kDa subunit of human C8, which is one of five components of the cytolytic membrane attack complex of complement (MAC). The three dimensional structure of recombinant C8γ was determined by X-ray diffraction to 1.2 Å resolution. The structure displays a typical lipocalin fold forming a calyx with a distinct binding pocket. When compared to other lipocalins, C8γ possesses a much deeper binding pocket allowing ligand access to a large hydrophobic cavity at the base of the calyx. Cys40 in C8γ, which forms the disulfide bond to C8γ, is located in a partially disordered loop near the opening of the calyx.; Chapter 3. Crystallographic investigations of C8γ in complex with lauric acid and Xe reveal key features of the ligand binding site located within the C8γ calyx. A detailed description of a hydrophobic cavity located within the deepest portions of the calyx is presented along with additional evidence for the Y83 Y131 gate.; Chapter 4. The X-ray crystal structure of human prostatic acid phosphatase (PAP) in complex with a phosphate ion has been determined at 2.4 Å resolution. Bound PEG molecules from the crystallization matrix have allowed the identification of channels within the protein that connect the active site with a trough located near the 2-fold axis at the dimer interface.; Chapter 5. The structure based design, synthesis and kinetics of a new tartrate based inhibitor, N-1,2-diphenylethyl-L-(+)-tartamate (pK_i = 6.7) is presented.; Chapter 6. Annexin V's calcium ion channel activity along with the calcium uptake in matrix vesicles is inhibited by Zn 2+, activated by ATP and modulated in a remarkable way by GTP. We have co-crystallized annexin V in complex with Ca2+, Zn 2+, ATP, and GTP and our preliminary findings are detailed.