Electronic Theses and Dissertations

Identifier

309

Angela Howard

Date

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Concentration

Organic Chemistry

Committee Chair

Abby L Parrill

Committee Member

Theodore J Burkey

Committee Member

Richard Petersen

Committee Member

Yongmei Wang

Abstract

Nucleotide pyrophosphatase/phophodiesterase 6 (NPP6) is a membrane anchored ectoenzyme initially expressed in the brain and kidney. It is the sixth member of the NPP superfamily. Currently, the superfamily consists of seven members, NPP1-NPP7. Each member was numbered according to the order in which it was associated with the family. The NPPsuperfamily hydrolyzes a wide range of substrates such as lipids, nucleotides, and their derivatives. NPP1, NPP2, and NPP3 hydrolyze nucleotides (and derivatives). NPP6 and NPP7 along with NPP2 hydrolyze phosphodiester bonds in lipids (and derivatives). Thus far, NPP4 and NPP5 have yet to be characterized in terms of which substrates they act upon or the activity which they posses. Many of the hydrolytic pathways of NPP enzymes lead to the formation of bioactive lipids that affect numerous pathological as well as physiological processes. This combined with their extracellular action makes them suitable targets for therapeutic intervention.NPP6 is expressed predominately in the kidneys where it plays a role in choline reabsorption. Choline is needed to maintain healthy kidneys; however over time choline degrades and the body does not replenish it. Therefore, structurally and functionally characterizating NPP6 is essential for potential kidney therapeutics.Here, we focused on testing and identifying more substrates for NPP6. Our results show that NPP6 is not a very promiscuous enzyme. Overall, NPP6 preferred hydrolyzing smaller substrates opposed to larger substrates. Like another member of the NPP superfamily, NPP7, NPP6 has lysophopholipase C activity. We identified lysophosphatidic acid 16:0 (LPC16:0) and para-nitrophenyl phosphocholine (pNPPC) as highly potent substrates for NPP6.Next, we focused on characterizing the structure and funciton of NPP6. Given that very little is known about the structure and function of NPP6, insight into this area may assist in the design of chemical probes of NPP6 physiological roles. All NPP superfamily members hydrolyze a wide range of substrates but exhibit unique substrate specificity profiles. Currently, little is known what defines the varying substrate specificity among the enzymes. To explore substrate specifying determinants for NPP6, we used computational modeling as a rational tool to guide selection of substrate recognition residues to experimentally mutate. We selected known substrates such as LPC16:0 and pNPPC and docked each into the NPP6 homlogy model. The experimental results validated the model suggesting the presence of a common binding pocket for the different substrates of NPP6. Furthermore, the experimental studies provide validation of our model for hydrogen bond interactions between the substrate and specific amino acid residues either during substrate recognition or during the catlytic process.The catalytic domain of the NPP superfamily has been associated with other sulfo- and phospho- coordinating metalloenzymes which include the alkaline phosphatase superfamily. Both the alkaline phosphatases and NPPs possess two divalent metal cations within their binding pocket and are inactivated by removal of the endogenous divalent metals. Until now, the role of the endogenous divalent metals has been unknown. Here, we show that the secondary structure is not affected by the presence or absence of the endogenous divalent metals; although, the catalytic activity is elmiinated upon overnight incubation with the metal chelator, EDTA. Attempts to restore the catalytic activity of NPP6 were made. Results showed that Ca2+ and Zn2+ restored almost complete 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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