Evidence that amino-acid residues are responsible for substrate synergism of locust arginine kinase.
A series of mutants were constructed to investigate the amino-acid residues responsible for the synergism in substrate binding of arginine kinase (AK). AK contains a pair of highly conserved amino acids (Y75 and P272) that form a hydrogen bond. In the locust (Locusta migratoria manilensis) AK, mutants in two highly conserved sites can cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. The Y75F and Y75D mutants showed strong synergism (Kd/Km=6.2-13.4), while in single mutants, P272G and P272R, and a double mutant, Y75F/P272G, the synergism was almost completely lost (Kd/Km=1.1-1.4). Another double mutant, Y75D/P272R, had characteristics similar to those of the wild-type enzyme. All these results suggest that the amino-acid residues 75 and 272 play an important role in regulating the synergism in substrate binding of AK. Fluorescence spectra showed that all mutants except Y75D/P272R displayed a red shift to different degrees. All the results provided direct evidence that there is a subtle relationship between the synergism in substrate binding and the conformational change.
Wu
Qing-Yun
QY
College of Life Science, Shandong Agricultural University, Tai'an, Shandong 271018, People's Republic of China.
Li
Feng
F
Wang
Xiao-Yun
XY
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001122:Arginine Kinase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015870:Gene Expression; D007700:Kinetics; D048188:Locusta migratoria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D001122:Arginine Kinase