&&ReWrAp:HEADERFOOTER:0:ReWrAp&&

Date of Award

8-2002

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Professor R.S. Gupta

Abstract

Adenosine kinase (AK) is a purine salvage enzyme which catalyzes the phosphorylation of the 5'-hydroxyl of adenosine via ATP. AK is a key enzyme which controls the intra and extracellular concentration of adenosine (Ado). Agents which inhibit the activity of AK have been found to attentuate cellular damage, demonstrating therapeutic utility in a variety of disease processes. In order to design inhibitors of AK with increased efficacy, a better understanding of enzyme activity is required. Previously a number of novel characteristics of mammalian AK had been discovered. It was shown that the activity of AK is influenced by the presence of pentavelent ions (PVI) such as inorganic phosphate (Pi), arsenate and vanadate. A detailed study of the influence of Pi on the kinetic parameters of Chinese hamster (CH) and beef liver AK was performed. These studies suggested that the Km (Ado) decreases and the Ki (Ado) increases asymptotically in the presence of increasing concentrations of Pi. Under the same conditions, the Vmax for activity increases hyperbolically. The effect of phosphate is not limited to the mammalian form of AK. Pi, arsenate and vandate were all found to have similar effects on AK from yeast, spinach and Leishmania donovani AK. PVI as well as the metabolite phoshoenol pyruvate were also found to stimulate the activity of the enzyme ribokinase (RK) from E. coli, which similar to AK, is a member of the PfkB family of carbohydrate kinases. Although AK and RK show little sequence similarity, the residues at the active site and the 3D structures of these two proteins are very similar. Based on sequence alignment of PfkB family members, we have identified a conserved sequence motif, NXXE, which based upon the available structural information appears to be involved in the binding of phosphate. To confirm and understand the role of this motif in Pi binding, the residues at the NXXE site were altered by site-directed mutagenesis and their effect on activity of the recombinant CH AK was examined. Though the residues at the NXXE site do not directly interact with substrate, nor the putative catalutic base, the resulting proteins were found to have greatly altered phosphate requirement, substrate inhibition characteristics and different magnesium requirements. In the AK structure, aspartic acid at position 316 presumed to act as the catalytic base. This residue was changed to aspargine and glumatic acid by mutagenesis. The resulting proteins were found to be nearly completely devoid of activity, confirming its critical role in AK activity. The amino acid sequence at the extreme N-terminus of AK has been found to exhibit the greatest variability within and among species, though the rest of the protein remains greatly conserved. To delineate the residues that are involved in the structural stability and activity of AK, systematic deletions of the residues from both the N- and C- terminus were performed, and the structure-activity relationships were examined. It was determined that the first 16 residues of CH AK can be removed without affecting activity. Removal of the next 11 residues in sequential decreases in enzyme stability and activity. These 11 residues are involved in the first ╬▓structure of the protein and are required for the stability of the tertiary structure. All residues at the C terminus were required for activity, and involved in a hydrogen-bonding network necessary for the stability of the ATP binding site. Thesestudies provide novel insight to the structure-activity relationship of mammalian AK as well as the PfkB family of enzymes. Our work has identified a site distal to the catalytic site, that is implicated in the PVI binding and catalytic effect. Further studies should be aimed at understanding how binding of PVI at this site influences the catalytic activity of AK. Development of inhibitors which bind to this site and modulate the activity of AK should prove very useful in this regard.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Biochemistry Commons

Share

COinS