The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability.
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 folding.
Guo
Qin
Q
Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Zhao
Feng
F
Guo
Shu-Yuan
SY
Wang
Xicheng
X
D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D047528:Stichopus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D014364:Tryptophan; D001122:Arginine Kinase; D019791:Guanidine