The X-ray structure of the Escherichia coli
aspartate transcarbamoylase with the bisubstrate analog
phosphonacetyl-l-aspartate (PALA) bound shows
that PALA interacts with Lys84 from an adjacent catalytic
chain. To probe the function of Lys84, site-specific mutagenesis
was used to convert Lys84 to alanine, threonine, and asparagine.
The K84N and K84T enzymes exhibited 0.08 and 0.29% of the
activity of the wild-type enzyme, respectively. However,
the K84A enzyme retained 12% of the activity of the wild-type
enzyme. For each of these enzymes, the affinity for aspartate
was reduced 5- to 10-fold, and the affinity for carbamoyl
phosphate was reduced 10- to 30-fold. The enzymes K84N
and K84T exhibited no appreciable cooperativity, whereas
the K84A enzyme exhibited a Hill coefficient of 1.8. The
residual cooperativity and enhanced activity of the K84A
enzyme suggest that in this enzyme another mechanism functions
to restore catalytic activity. Modeling studies as well
as molecular dynamics simulations suggest that in the case
of only the K84A enzyme, the lysine residue at position
83 can reorient into the active site and complement for
the loss of Lys84. This hypothesis was tested by the creation
and analysis of the K83A enzyme and a double mutant enzyme
(DM) that has both Lys83 and Lys84 replaced by alanine.
The DM enzyme has no cooperativity and exhibited 0.18%
of wild-type activity, while the K83A enzyme exhibited
61% of wild-type activity. These data suggest that Lys84
is not only catalytically important, but is also essential
for binding both substrates and creation of the high-activity,
high-affinity active site. Since low-angle X-ray scattering
demonstrated that the mutant enzymes can be converted to
the R-structural state, the loss of cooperativity must
be related to the inability of these mutant enzymes to
form the high-activity, high-affinity active site characteristic
of the R-functional state of the enzyme.