Direct thermodynamic and kinetic investigations
of the binding of nucleotides to the nucleoside monophosphate
(NMP) site of NMP kinases have not been possible so far
because a spectroscopic probe was not available. By coupling
a fluorescent N-methylanthraniloyl- (mant) group to the
β-phosphate of CDP via a butyl linker, a CDP analogue
[(Pβ)MABA-CDP] was obtained that
still binds specifically to the NMP site of UmpKdicty,
because the base and the ribose moieties, which are involved
in specific interactions, are not modified. This allows
the direct determination of binding constants for its substrates
in competition experiments.