AUTOPROTEOLYSIS OF N-TERMINAL NUCLEOPHILE HYDROLASE

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DESCRIPTION (Adapted from abstract): Lysosomal glycosylasparaginase hydrolyzes the amide bond joining carbohydrate to protein in Asn-linked glycoproteins. This enzyme joins the proteasome (Lowe et al., 1995; Groll et al., 1997) and penicillin acylase (Duggleby et al., 1995) as a class of amidases that catalytically use a processed N-terminal threonine or serine as both a polarizing base and a nucleophile. Another intriguing aspect of this enzyme is that a single chain precursor is processed by intramolecular autoproteolysis to yield the conserved N-terminal threonine and an active amidase (Guan et al., 1996). With crystals in hand, Dr. Guo proposes to determine the crystal structures of glycosylasparaginase precursor (proenzyme) to elucidate the mechanism of this intramolecular autoproteolysis, and activation process of enzyme activity. Based on structural geometry and evolutionarily conserved sequence, the residues appearing to be important for autoproteolysis will be selected for site-directed mutagenesis. Mutant enzymes will then be subject to kinetic analysis, and/or further physical studies by circular dichroism (CD) or crystallography. Dr Guo will further investigate the role of dimerization in the intramolecular autoproteolysis. He has also crystallized the mutant proteins of reduced activity in their mature (autocleaved) form. By applying cryocrystallography techniques, he will attempt to stabilize the enzyme-substrate (or inhibitor) complexes for x-ray structure determination, to allow a more detailed examination of the enzymatic mechanism. The enzymatic activities and enzyme activation are central to much of physiology. Dr. Guo's work addresses the mechanisms of function at atomic resolution, and should contribute to an increased understanding of the essential biological processes. The broad, long-term objectives of this research plan are to understand the molecular basis of enzymatic mechanisms, as well as protein splicing. The tools employed are x-ray crystallography, CD, molecular biology, and protein chemistry.