Associate Provost for Research Boston University Medical Campus
NIDDK - National Institute of Diabetes & Digestive & Kidney Diseases	Research Resources

Abstract

Abstract: 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.

Thesaurus Terms:
asparaginase, enzyme activity, enzyme mechanism, enzyme structure, proteolysis, zymogen
active site, amidohydrolase, chemical cleavage, chemical kinetics, dimer, enzyme substrate complex, glycosidase, proteasome
X ray crystallography, circular dichroism, site directed mutagenesis

Institution:	BOSTON UNIVERSITY MEDICAL CAMPUS
	715 ALBANY ST, 560
	BOSTON, MA 021182394
Fiscal Year:	2004
Department:	PHYSIOLOGY AND BIOPHYSICS
Project Start:	01-AUG-1999
Project End:	31-JUL-2004
ICD:	NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
IRG:	BBCB