Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.
The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.
Bao
Jie
J
Biochemical Engineering Program, University of California, One Shields Ave., Davis, CA 95616, USA.
Ryu
Dewey D Y
DD
D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D003838:Deoxyadenine Nucleotides / Q000096:biosynthesis*; D003848:Deoxyguanine Nucleotides / Q000096:biosynthesis*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D011770:Pyruvate Kinase / Q000737:chemistry
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; C029603:deoxyguanosine triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate