Site-specific mutations of conserved residues in the phosphate-binding loop of the Arabidopsis UMP/CMP kinase alter ATP and UMP binding.
All eukaryotic UMP/CMP kinases contain a glycine-rich sequence GGPG(S/A)GK at the N-terminus. This sequence is homologous to the conserved sequence GXXGXGK found in other ATP-binding proteins. To study the role of this conserved sequence in Arabidopsis UMP/CMP kinase, five conserved residues were mutated by site-directed mutagenesis to generate seven mutant enzymes: G21A, G22A, G24A, G26A, K27R, K27M, and K27E. The G21A and G26A mutants were degraded during the purification phase and were thus unable to be purified. Kinetic studies on the other mutants, when compared to studies on the wild-type enzyme, revealed that this sequence is important for ATP binding and enzyme catalysis. All mutants had a decreased kcat/KATPm value. The G22A and G24A mutants had about half of the kcat value of wildtype and 3.9-fold and 3.3-fold increases in KATPm values, respectively. The kcat/KATPm values in the K27M and K27E mutants were changed significantly and decreased by 1000-fold and 2600-fold, respectively. The removal of the terminal positive charge of Lys27 in the K27M and K27E mutants resulted in 20% of the kcat value of wildtype. However, both mutants had a remarkable increase in KATPm value by 241-fold and 552-fold, respectively. Therefore, the positive charge of Lys27 plays an important role on both ATP binding and enzyme catalysis. Interestingly, the results also showed that the mutations that affected ATP binding also had an effect on UMP binding.
Zhou
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Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa, 50011, USA.
Thornburg
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R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D017360:Arabidopsis; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D017433:Protein Structure, Secondary; D014542:Uridine Monophosphate / Q000378:metabolism*
D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase