Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain
V. Serre et al., Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain, J BIOL CHEM, 274(34), 1999, pp. 23794-23801
The first two steps of the de novo pyrimidine biosynthetic pathway in Sacch
aromyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded
by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate s
ynthetase (CPSase) and aspartate transcarbamoylase (ATCase) function indepe
ndently, there are interdomain interactions uniquely associated with the mu
ltifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP
. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSa
se domain where it is synthesized to the ATCase domain where it is used in
the synthesis of carbamoyl aspartate. To better understand these processes,
a recombinant plasmid was constructed that encoded a protein lacking the a
midotransferase domain and the amino half of the CPSase domain, a 100-kDa c
hain segment. The truncated complex consisted of the carboxyl half of the C
PSase domain fused to the ATCase domain via the pDHO domain, an inactive di
hydroorotase homologue that bridges the two functional domains in the nativ
e molecule. Not only was the "half CPSase" catalytically active, but it was
regulated by UTP to the same extent as the parent molecule. In contrast, t
he ATCase domain was no longer sensitive to the nucleotide, suggesting that
the two catalytic activities are controlled by distinct mechanisms. Most r
emarkably, isotope dilution and transient time measurements showed that the
truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase
reaction is much less sensitive than the parent molecule to the ATCase bisu
bstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence t
hat the endogenously produced carbamoyl phosphate is sequestered and channe
led to the ATCase active site.