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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053893-02
Application #
6177611
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Laughlin, Maren R
Project Start
1999-08-01
Project End
2003-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
2
Fiscal Year
2000
Total Cost
$174,012
Indirect Cost
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Sun, Yixin; Guo, Hwai-Chen (2008) Structural constraints on autoprocessing of the human nucleoporin Nup98. Protein Sci 17:494-505
Wang, Yeming; Guo, Hwai-Chen (2007) Crystallographic snapshot of a productive glycosylasparaginase-substrate complex. J Mol Biol 366:82-92
Xu, Qian Steven; Kucera, Rebecca B; Roberts, Richard J et al. (2004) An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site. Structure 12:1741-7
Wang, Xingtai; Qian, Xiaofeng; Guo, Hwai-Chen et al. (2003) Heat shock protein 90-independent activation of truncated hepadnavirus reverse transcriptase. J Virol 77:4471-80
Wang, Yeming; Guo, Hwai-Chen (2003) Two-step dimerization for autoproteolysis to activate glycosylasparaginase. J Biol Chem 278:3210-9
Qian, Xiaofeng; Guan, Chudi; Guo, Hwai-Chen (2003) A dual role for an aspartic acid in glycosylasparaginase autoproteolysis. Structure 11:997-1003
Xu, Q; Buckley, D; Guan, C et al. (1999) Structural insights into the mechanism of intramolecular proteolysis. Cell 98:651-61
Cui, T; Liao, P H; Guan, C et al. (1999) Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase. Acta Crystallogr D Biol Crystallogr 55:1961-4