Human soluble calcium-activated nucleotidase 1 (hSCAN-1) represents a new family of apyrase enzymes, the Calcium Activated Nucleotidases (CANs), which catalyze the hydrolysis of various di- and tri-nucleotides. Biologically, apyrases are involved in the regulation of extra-cellular purinergic and pyrimidinergic signaling, which associates their functionality with homeostatic processes such as the maintenance of cellular calcium levels. The enhanced expression of hSCAN-1 itself has recently been associated with stress-related processes (e.g. unfolded protein response), as well as specific disease states (e.g. Desbuquois dysplasia, prostate cancer). From a structural and functional perspective, the CANs differ from other well-characterized nucleotide-phosphate-binding enzymes. We have found that they posses a unique phosphate-binding motif, which lacks the common P-loop and is dominated by ionizable residues. Catalytically, they display highly specific calcium dependence, distinct from the cationic promiscuity of the NTPDase apyrase family. Motivated by the initial success in the redesign of hSCAN-1 into a potentially therapeutic anti-coagulant, we are the first to computationally investigate the active site structure, reactivity, and catalytic mechanism of a member from this novel phosphoryl transfer family. Supported by molecular dynamics simulations, we have revealed a highly symmetric, yet previously unrecognized, second calcium-binding site, which is the key to the family's exclusive calcium activation and explains the unusual, experimentally observed, sigmoidal relationship between calcium concentration and hSCAN-1 activity. We present how the ionizable nature of this binding site can account for additional experimental observations: a narrow pH range for optimal hSCAN-1 activity and a moderate enzymatic inhibition in the presence of sodium. Supported by ab initio QM/MM molecular dynamics simulations, we propose a detailed phosphoryl transfer mechanism for hSCAN-1, which relies upon the loose 8-9-fold coordination around this catalytic calcium. This coordination structure includes two protonated ionizable residues, Asp44 and Asp114, that properly orient the nucleophilic water. A third protonated Asp46 donates its proton, through the substrate, to neutralize the bridging oxygen leaving group. In light of our theoretical results and interpretation, we propose specific mutagenesis intended to enhance the rate of nucleotide hydrolysis and combined mutagenesis/kinetics experiments designed to empirically support our findings.
|Commitee:||Broyde, Suse, Kallenbach, Neville, Mezei, Mihaly, Tuckerman, Mark|
|School:||New York University|
|School Location:||United States -- New York|
|Source:||DAI-B 74/01(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Biophysics|
|Keywords:||Apyrase, Calcium activated nucleotidases, Hscan-1, Molecular dynamics, Phosphoryl transfer, Qmmm|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be