Functional and structural studies on glycogenin

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

William J. Whelan, Committee Chair


The synthesis of glycogen, the body's major energy storage molecule, is dependent upon a protein primer that represents the biosynthetic origin of the polysaccharide. Glycogenin is the autocatalytic, self-glucosylating protein that primes the synthesis of glycogen by generating, on itself, the maltosaccharide chain which becomes the substrate for glycogen synthase and branching enzyme. After glucosylation of a tyrosine residue at position 194 on the polypeptide by a mechanism unknown until now, glycogenin autocatalytically extends its glucan chain to form malto-octaose, using uridine diphosphate glucose. This chain then serves as the base for further polymerization by the synthesizing enzymes. A full length cDNA clone encoding rabbit muscle glycogenin was isolated. The bacterial expression of the recombinant protein yielded an already partially glucosylated product capable of incorporating, in vitro, additional glucose. A few aspects of the enzymatic activity of glycogenin were revealed. In addition to transferring glucose residues from UDPglucose, this protein concomitantly hydrolyses the glucose donor at a rate comparable with that of self-glucosylation. In regard to specificity, all pyrimidine glucose nucleotides are competent substrates both for self- and transglucosylation, to varying degrees. The analysis of mutant proteins revealed the importance of structural features pertaining to the glucosyl transfer and accepting capacity of glycogenin. The aromatic moiety of Tyr194 at the glycosidic linkage is crucial for the ability of the protein to receive transferred glucose. When Tyr194 was substituted by a threonine or phenylalanine, the mutant molecules could not self-glucosylate. No other site in the molecule can anchor carbohydrate. However, the glucose acceptor capacity of the protein is completely independent of its catalytic activity. Autocatalytically inactive mutant glycogenin was found to be an active transglucosylase when offered an appropriate alternative glucose acceptor substrate. The substitution of the tyrosine residue at position 194 did not cause a significant departure from the wild-type kinetics and enzymatic properties; it simply abolished the glucose accepting capacity of the protein. Finally, the autocatalytic nature of the addition of the first glucose residue to Tyr194 was elucidated by generating carbohydrate-free protein by two different methods and assaying for the formation of the maltosaccharide chain.


Biology, Molecular; Chemistry, Biochemistry

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