Studies on yeast nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). IV. Steady-state kinetic properties with thymidine nucleotides (including 3'-azido-3'-deoxythymidine analogues).
A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.
Kuby
S A
SA
Department of Biochemistry, University of Utah, Salt Lake City.
Fleming
G
G
Alber
T
T
Richardson
D
D
Takenaka
H
H
Hamada
M
M
D000227:Adenine Nucleotides / Q000378:metabolism; D001667:Binding, Competitive; D007700:Kinetics; D008956:Models, Chemical; D010770:Phosphotransferases / Q000737:chemistry*; D012440:Saccharomyces / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism; D015215:Zidovudine / Q000378:metabolism
D000227:Adenine Nucleotides; D013942:Thymine Nucleotides; D015215:Zidovudine; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase