As an alternative method to study the heterotropic
mechanism of Escherichia coli aspartate transcarbamoylase,
a series of nucleotide analogs were used. These nucleotide
analogs have the advantage over site-specific mutagenesis
experiments in that interactions between the backbone of
the protein and the nucleotide could be evaluated in terms
of their importance for function. The ATP analogs purine
5′-triphosphate (PTP), 6-chloropurine 5′-triphosphate
(Cl-PTP), 6-mercaptopurine 5′-triphosphate (SH-PTP),
6-methylpurine 5′-triphosphate (Me-PTP), and 1-methyladenosine
5′-triphosphate (Me-ATP) were partially synthesized
from their corresponding nucleosides. Kinetic analysis
was performed on the wild-type enzyme in the presence of
these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP,
and SH-PTP each activate the enzyme at subsaturating concentrations
of l-aspartate and saturating concentrations of
carbamoyl phosphate, but not to the same extent as does
ATP. These experiments suggest that the interaction between
N6-amino group of ATP and the backbone of the
regulatory chain is important for orienting the nucleotide
and inducing the displacements of the regulatory chain
backbone necessary for initiation of the regulatory response.
Me-PTP and Me-ATP also activate the enzyme, but in a more
complex fashion, which suggests differential binding at
the two sites within each regulatory dimer. The purine
nucleotides GTP, ITP, and XTP each inhibit the enzyme but
to a lesser extent than CTP. The influence of deoxy and
dideoxynucleotides on the activity of the enzyme was also
investigated. These experiments suggest that the 2′
and 3′ ribose hydroxyl groups are not of significant
importance for binding and orientation of the nucleotide
in the regulatory binding site. 2′-dCTP inhibits
the enzyme to the same extent as CTP, indicating that the
interactions of the enzyme to the O2-carbonyl
of CTP are critical for CTP binding, inhibition, and the
ability of the enzyme to discriminate between ATP and CTP.
Examination of the electrostatic surface potential of the
nucleotides and the regulatory chain suggest that the complimentary
electrostatic interactions between the nucleotides and
the regulatory chain are important for binding and orientation
of the nucleotide necessary to induce the local conformational
changes that propagate the heterotropic effect.