Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino -5-bromouracil and 1-(8-phosphonooetyl)-7-deazaxanthine inhibitors of Esche richia coli thymidine (dThd) phosphorylase (TPase), The structure of the co mpounds was rationally designed based on the available crystal structure co ordinates of bacterial TPase, These inhibitors reversibly inhibited TPase, Kinetic analysis revealed that the compounds inhibited TPase in a purely co mpetitive or mixed fashion not only when dThd, but also when inorganic phos phate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhib itors of the enzyme in the presence of variable Pi concentrations while bei ng competitive or mixed with respect to thymine as the natural substrate. O ur kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine /purine derivatives are able to function as multisubstrate inhibitors of TP ase, interfering at two different sites (dThd(Thy)- and phosphate-binding s ite) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they shoul d be considered as lead compounds for the development of mechanistically no vel type of TPase inhibitors. (C) 2000 Federation of European Biochemical S ocieties; Published by Elsevier Science B.V. All rights reserved.