Electronic Theses and Dissertations

Identifier

141

Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Concentration

Analytical Chemistry

Committee Chair

Daniel L. Baker

Committee Member

Richard L. Peterssen

Committee Member

Theodore J. Burkey

Committee Member

Yongmei Wang

Abstract

Nucleotide pyrophosphatase/phosphdiesterase (NPP) 7 , also known as alkaline sphingomyelinase (Alk-SMase) is a membrane-anchored ectoenzyme initially discovered in the intestinal tract in 1969. It is the most recently identified NPP isoform.The NPP family of enzymes currently consists of seven members, NPP1-7, numbered according to the order in which they were first associated with the NPP family.NPP enzymes hydrolyze a wide range of substrates. NPP1 and 3 are nucleotide pyrophosphatases that cleave inorganic phosphate from nucleatides and their derivatives. In contrast, NPP2, NPP6 and NPP7 are phosphodiesterases that hydrolyze phosphodiester bonds in lysolipid substrates and their derivatives. NPP4 and NPP5 are yet to characterized in terms of substrate preference and biological function. Numerous lysolipid substrates have been shown to be hydrolyzed by NPP2 and NPP6 but only three main lysolipid substrates have been previously reported for NPP7. One of the goals of this work was to characterize the substrate preference of NPP7 through exhaustive kinetic means. Our results show that NPP7, like NPP2 and NPP6, hydrolyzes numerous choline-containg substrates including glycerolipids, lysophosphatidylcholine, platelet activating factor, lyso platelet activating factor and sphingolipids, sphingomyelin and sphingosylphosphorylcholine. A synthetic small molecule, para-nitrophenyl phosphocholine was also identified as a substrate for NPP7. Most hydrolytic activities of NPP isoforms lead to formation of bioactive lipids that affect numerous physiological and pathological processes. This, coupled with their extracellular activity, makes them attractive targets for therapeutic intervention. A major bottleneck to their utilization is that very little is known about their structure and function. Although NPP enzymes hydrolyze a wide range of substrates and sometimes share a common substrate, they exhibit different substrate preference profiles. The factore influencing these different preferences have not been investigated. Our second goal therefore was to employ computational modeling as a rational tool to guide experimental procedures in exploring NPP7 substrate specificity determinants. Our modeling results suggest a common binding pocket for the different substrate groups and the presence of essential non covalent interactions between the substrates and specific amino acid residues. Like alkaline phosphates, NPP isoforms require divalent metal cations for catalytic activity but it is not clearly known whether the divalent metals play both a function and structural role within the enzyme. Our third goal was therefore to explore the role played by divalent metal cation s in NPP7. Our results indicate that the secondary structure of NPP7 is not altered in the absence or presence of divalent metal cations. However, its catalytic function is eliminated with prolonged exposure to sufficient mounts of metal chelators. The current work significantly advances our understanding of the NPP7 in terms of preferred substrates, the amino acid determinants that underlie these preferences and the role of divalent catalytic site metal ions in the structure and function of the enzyme .

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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