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STUDIES OF RAT LIVER 4-HYDROXY-2-KETOGLUTARATE ALDOLASE AND 4-HYDROXY-2-KETOGLUTARATE METABOLISM
Abstract
The goal of this research was to understand the kinetic mechanism and other characteristics of rat liver 4-hydroxy-2-ketoglutarate aldolase, and its role in the metabolism of 4-hydroxy-2-ketoglutarate. The enzyme catalyzes a reversible retro-aldol cleavage of 4-hydroxy-2-ketoglutarate. The condensations of pyruvate or oxaloacetate with glyoxylate to form 4-hydroxy-2-ketoglutarate, both catalyzed by the enzyme, were found to proceed via an ordered reaction mechanism. Studies using chemical modification were performed to elucidate the essential amino acid residues present in the active site of the enzyme. The studies indicated the presence of an arginyl residue and a sulfhydryl residue in the active site. The latter residue was found to be located in close proximity to the Schiff base-forming lysyl residue of the active site. The condensation of pyruvate and glyoxylate catalyzed by the aldolase was stimulated by the addition of 4-hydroxy-2-ketoglutarate, the product of the reaction. This stimulation might be due to the allosteric regulation of the enzyme by 4-hydroxy-2-ketoglutarate. However, the involvement of a mechanism resulting in the exchange of glyoxylate, involving the rapid cleavage and synthesis of 4-hydroxy-2-ketoglutarate, would also account for the stimulation. The aldolase will catalyze the condensation of formaldehyde and pyruvate to form 4-hydroxy-2-ketobutyrate. Formaldehyde was found to be the only aldehyde of those tested which would replace glyoxylate in a catalyzed condensation with pyruvate. Similarly, other pyruvate analogs were tested for their ability to undergo a catalyzed condensation with formaldehyde, but the reaction was specific for pyruvate only. Experiments were performed in an attempt to isolate the enzyme 2-ketoglutarate 4-hydroxylase. The existence of this enzyme has previously been postulated, but has not yet been established. An enzyme which exhibits similar characteristics to those of the postulated hydroxylase was isolated from cytosolic extracts of suckling rat liver. The characteristics and physical properties of this enzyme were studied. The possible role of the enzyme in the metabolism of 4-hydroxy-2-ketoglutarate and gluconeogenesis is discussed.
Subject Area
Biochemistry
Recommended Citation
HRUBEY, TODD WARREN, "STUDIES OF RAT LIVER 4-HYDROXY-2-KETOGLUTARATE ALDOLASE AND 4-HYDROXY-2-KETOGLUTARATE METABOLISM" (1987). ETD collection for University of Nebraska-Lincoln. AAI8803998.
https://digitalcommons.unl.edu/dissertations/AAI8803998