Biochemistry, Department of

 

Date of this Version

Spring 4-19-2011

Comments

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Joseph J. Barycki. Lincoln, Nebraska: May, 2011

Copyright 2011 Melanie M. Neely Willis

Abstract

Cellular detoxification allows for the maintenance of cellular homeostasis and prevention of abnormal cell growth by clearing harmful xenobiotics and endobiotics. After oxygenation by phase I enzymes, phase II enzymes such as glucuronosyltransferases and glutathione-s-transferases conjugate a small molecule to the compound, marking it for subsequent export. Many up-stream enzymes are also essential to cellular detoxification by supplying the small compounds for conjugation. These up-stream enzymes include UDP-glucose dehydrogenase, which synthesizes UDP-glucuronate, and glutamate cysteine ligase, which catalyzes the first and rate-limiting step in the synthesis of glutathione.
UDP-glucose dehydrogenase (UGDH) is an important enzyme in human development and in the progression of many types of human epithelial cancers. Recently, mutations in UGDH were identified that are associated with congenital heart defects and cause a shift from a hexameric to a dimeric state. These clinical mutants, along with two engineered dimer mutants were used to examine differences in UGDH function resulting from loss of hexameric structure. The dimer mutants exhibited near wild-type activity in vitro, and significant differences in UDP-glucuronate levels were not observed in HEK 293 cells. Despite this, the phenotype of development defects associated with the UGDH clinical mutants is at least partially explained by a reduction in protein stability.
Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM). Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC and cellular glutathione levels were determined to be significantly lower in the mutants relative to wild-type. In an S. cerevisiae model system, mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM, while the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC.