Kinetic mechanism of deoxyadenosine kinase from Mycoplasma determined by surface plasmon resonance technology.
Surface plasmon resonance (SPR) detection technology was employed to investigate the kinetic mechanism of deoxyadenosine kinase from Mycoplasma mycoides ssp. mycoides SC. In our experimental approach, the enzyme was attached to the sensor surface, the reactants were injected in the mobile phase, and the product-enzyme complex formation was measured using the fact that the rate of product formation exceeds that of its dissociation. The pre-steady-state analysis of deoxyguanosine phosphorylation showed the presence of a burst phase, which is consistent with product dissociation being a rate-limiting step. High activity of the immobilized enzyme was demonstrated by analyzing the reaction mixture eluted from the chip and by determining the Michaelis-Menten constants for several phosphate acceptors (e.g., deoxyadenosine) and phosphate donors (e.g., ATP) using SPR detection. These values were in good agreement with those reported previously [Wang, L. et al. (2001) Mol. Microbiol. 42, 1065-1073]. The bisubstrate initial rate pattern obtained was characteristic of a sequential kinetic mechanism. Because in the method applied here it is the mass change on the surface that is monitored, a new mathematical approach to interpreting product inhibition experiments was proposed. According to that approach, product inhibition studies, supported by product binding experiments, indicated that the reaction mechanism was of Bi Bi sequential ordered type, involving the formation of a ternary complex, in which ATP and deoxyadenosine bound sequentially, followed by a transfer of the phosphate group, and an ordered release of products with ADP dissociating before dAMP.
Pol
Ewa
E
Biacore AB, SE-754 50 Uppsala, Sweden. ewa.pol@biacore.com
Wang
Liya
L
D000244:Adenosine Diphosphate / Q000378:metabolism; D001667:Binding, Competitive; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009176:Mycoplasma mycoides / Q000201:enzymology*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D003838:Deoxyadenine Nucleotides; D000244:Adenosine Diphosphate; C012873:2'-deoxy-5'-adenosine monophosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020005:deoxyadenosine kinase