Date of Award:

5-2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Alvan C. Hengge

Committee

Alvan C. Hengge

Committee

Bradley S. Davidson

Committee

Sean J. Johnson

Committee

Daren P. Cornforth

Committee

Lisa M. Berreau

Abstract

Protein phosphorylation is as an important regulatory mechanism that controls a large number of physiologically important cellular processes. Proteins are phosphorylated on aliphatic (serine and threonine) as well aromatic (histidine and tyrosine) residues predominantly located on sterically accessible loops. Phosphorylation affects three- dimensional structures of proteins resulting in conformational changes which activate or deactivate enzymatic functions. Because of the ability of protein phosphorylation to modulate a multitude of diverse cellular processes, it became an appealing and promising target for drug therapy.

Attachment of phosphate is accomplished by kinases, whereas the removal of the phosphoryl group is performed by protein phosphatases. Traditionally, protein kinases have been studied more intensely than protein phosphatases due to the earlier belief that they confer fine regulation to protein phosphorylation, whereas protein phosphatases act to nonspecifically remove phosphate groups. In recent years the protein phosphatases were subjected to thorough analysis to reveal a number of diverse properties which formed the basis for phosphatase classification. The family of protein tyrosine phosphatses (PTPs) represents the largest group of protein phosphatases known today.

We focused our study on the 150 amino acids VHZ phosphatase, which was originally classified as a dual-specificity enzyme which can dephosphorylate aliphatic (phospho serine, phospho threonine) and aromatic (phospho tyrosine) residues. We solved a crystal structure of VHZ refined to 1.1 Å resolution and performed an extensive structural, biological and kinetic analysis which revealed that this enzyme is not a dual specificity phosphatase. Activity screening with 360 biologically relevant peptides indicated strict phospho-tyrosine specificity and provided important information about its physiological substrates. Mechanistic studies using steady-state and pre-steady state kinetics, as well as inhibition studies and kinetic isotope effects (KIE) explain the significantly reduced catalytic parameters of VHZ, and contribute to our understanding of the PTP catalytic mechanism in general.

Checksum

89e7bc454032b8a60f2f60783ef0f57e

Download

Share

COinS

Copyright for this work is retained by the student. If you have any questions regarding the inclusion of this work in the Digital Commons, please email us at .