Characterization of band 3 interactions with glycolytic enzymes and preliminary NMR investigations of KP4 toxin
Abstract
The purpose of the present study was to investigate the interaction of human erythroctyte band 3 with glycolytic enzymes and to determine the structure of KP4 toxin by NMR Spectroscopy. A fifteen residue peptide of band 3 (B3P) containing the glycolytic enzyme binding region was used in this study. Presented in Chapter 2 were competitive inhibition assays (K$\rm\sb{I}=52\ \mu$M) and stoichiometric measurements performed with B3P when complexed with rabbit muscle aldolase. Chapter 3 describes the three-dimensional NMR structure of B3P when bound to aldolase determined by utilizing the exchange-transferred nuclear Overhauser effect (ET-NOE). Two major structural components of B3P in the bound state are described: a loop-like structure about Tyr 8; a hydrophobic cluster was formed in which the aromatic ring of Tyr 8 was sandwiched between methyl groups of Leu 4 and Met 12. Molecular modeling the bound structure of B3P in the active site region of aldolase is presented in Chapter 4. It was shown that the loop-like structure of B3P described in Chapter 3 forms a complementary fit both sterically and electrostatically with the bowl-shaped active site of aldolase. Since phosphorylation of Tyr 8 has been shown to disrupt the band3:glycolytic enzyme complex, a phosphate was modeled onto Tyr 8 within the B3P structure docked in the aldolase active site. The interaction energy was calculated and intramolecular electrostatic repulsion was shown to be the likely cause in disrupting the band 3:glycolytic enzyme complex. In Chapter 5, aldolase inhibition assays with methoxytyrosyl B3P showed that steric repulsion was unlikely to prevent the band 3:glycoltyic enzyme complex. Furthermore, phosphotyrosyl B3P (pB3P) was found to be a more potent inhibitor than B3P when the opposite was expected. This phenomenon can be explained in Chapter 6 in which evidence for a novel phosphotyrosine binding domain may reside somewhere in the cytoplasmic domain of band 3 and may be important in glycolytic regulation in the red cell. Finally, disulfide bond determination and preliminary NMR structural investigations of KP4 toxin are described in Chapter 7.
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biophysics|Biochemistry|Pharmacology
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