Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase.

The Journal of Biological Chemistry
Y LiuN N Aronson

Abstract

Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affini...Continue Reading

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Citations

Feb 7, 2012·Proceedings of the National Academy of Sciences of the United States of America·Andrew R BullerCraig A Townsend
Feb 6, 2013·Proceedings of the National Academy of Sciences of the United States of America·Andrew R Buller, Craig A Townsend
Feb 20, 2007·Applied and Environmental Microbiology·Eric HüfnerChristian Hertel
May 30, 2009·The FEBS Journal·Diana ZhiryakovaNicolina Stambolieva
Dec 13, 2006·Journal of Molecular Biology·Yeming Wang, Hwai-Chen Guo
Aug 31, 2010·Journal of Molecular Biology·Yeming Wang, Hwai-Chen Guo
Aug 31, 2000·Annual Review of Biochemistry·H Paulus
Aug 9, 2003·Structure·Xiaofeng QianHwai-Chen Guo
Mar 23, 2019·Protein Science : a Publication of the Protein Society·Suchita Pande, Hwai-Chen Guo
May 16, 2000·The Journal of Biological Chemistry·A F KisselevA L Goldberg
Dec 1, 2011·The Journal of Biological Chemistry·Marcin DembekNeil F Fairweather

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