Chemistry, Department of


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A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor David B. Berkowitz. Lincoln, Nebraska: May, 2011
Copyright 2011 Leah Zohner


Pyridoxal phosphate (PLP) dependent enzymes have the ability to manipulate amino acid substrates, serving variously as (i) racemases, transaminases, and beta- or gamma eliminases (all involving Cα-H bond cleavage); (ii) decarboxylases (Cα-CO2- bond cleavage), or (iii) retroaldolases (Cα-Cβ bond cleavage). Dunathan posited that stereoelectronics govern the key C-X bond cleavage step across the class of PLP enzymes; namely by aligning the scissile bond of the substrate with the extended pi system of the substrate-PLP imine that bond is weakened. A mechanistic understanding of electron flow in this enzymatic class has motivated many groups, including the Berkowitz group, to develop mechanism-based enzyme inactivators for specific PLP enzymes. Most relevant to this thesis is the finding that L-alpha-(2’Z- fluoro)vinyllysine, designed as a “suicide substrate” is, indeed, an efficient irreversible inactivator (t1/2 ~ 3 min, Ki ~ 100 uM, partition ratio ~ 16) of lysine decarboxylase from Hafnia alvei (K. R Karukurichi et al. J. Am. Chem. Soc. 2007, 129, 258-9) while the D-antipode is a slow substrate.

This thesis is motivated by the desire to better understand this interesting result at the molecular level. Described is a streamlined protocol for the purification of this useful model enzyme from the native source, Hafnia alvei. The ultimate goal is prepare homogeneous protein of sufficient quality and quantity to permit its successful crystallization to yield diffraction quality crystals. This thesis details and documents an improved purification procedure of LDC, as well as presents preliminary data toward its crystallization. The thesis will also review related key precedents in the field, both for the successful mechanism based inactivation of PLP dependent enzymes, and for the structural inactivation, principally involving with protein crystallography.

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