Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase.
The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.
Huang
H S
HS
School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan.
Ito
K
K
Kabashima
T
T
Yoshimoto
T
T
D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D005991:Glycerol Kinase / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013824:Thermus / Q000737:chemistry*
D018698:Glutamic Acid; D005991:Glycerol Kinase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin