The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability

Biochimie
Qin GuoXi-Cheng Wang

Abstract

Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein f...Continue Reading

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Citations

Apr 30, 2008·Bioprocess and Biosystems Engineering·L CostaJ M S Cabral
Mar 4, 2014·International Journal of Biological Macromolecules·Hong-Li GengKai-Lin Xu
Jun 8, 2011·International Journal of Biological Macromolecules·Na LiuJi-Cheng Pan
Jul 25, 2009·International Journal of Biological Macromolecules·Qing-Yun WuXiao-Yun Wang
Aug 16, 2008·International Journal of Biological Macromolecules·Qing-Yun WuXiao-Yun Wang
Dec 12, 2007·Insect Biochemistry and Molecular Biology·Qing-Yun WuXiao-Yun Wang
Nov 13, 2007·International Journal of Biological Macromolecules·Tao-Tao Liu, Xi-Cheng Wang
Sep 4, 2007·International Journal of Biological Macromolecules·Wen-Jing ZhuXiao-Yun Wang
Jul 26, 2005·International Journal of Biological Macromolecules·Zhao FengWang Xicheng
May 10, 2011·Archives of Biochemistry and Biophysics·Qing-Yun WuZheng Jun Chen
Apr 14, 2016·Journal of Bioenergetics and Biomembranes·Alonso A Lopez-ZavalaKarina D Garcia-Orozco
Jan 1, 2012·Computational and Structural Biotechnology Journal·Kerstin Steiner, Helmut Schwab
Sep 6, 2011·International Journal of Biological Macromolecules·Hua-Rong WangXiao-yun Wang

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