Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 angstrom
L. Jin et al., Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 angstrom, PROTEINS, 37(4), 1999, pp. 729-742
A high-resolution structure of Escherichia coli aspartate transcarbamoylase
has been determined to 2.1 Angstrom; resolution in the presence of the bis
ubstrate analog N-phosphonacetyl-L-aspartate (PALA). The structure was refi
ned to a free R-factor of 23.4% and a working R-factor of 20.3%. The PALA m
olecule is completely saturated with interactions to side chain and backbon
e groups in the active site, including two interactions that are contribute
d from the 80s loop of the adjacent catalytic chain, The charge neutralizat
ion of the bound PALA molecule (and presumably the substrates as well) indu
ced by the electrostatic field of the highly positively charged active site
is an important factor in the high binding affinity of PALA and must be im
portant for catalysis, The higher-resolution structure reported here depart
s in a number of ways from the previously determined structure at lower res
olution. These modifications include alterations in the backbone conformati
on of the C-terminal of the catalytic chains, the N- and C-termini of the r
egulatory chains, and two loops of the regulatory chain. The high-resolutio
n of this structure has allowed a more detailed description of the binding
of PALA to the active site of the enzyme and has allowed a detailed model o
f the tetrahedral intermediate to be constructed. This model becomes the ba
sis of a description of the catalytic mechanism of the transcarbamoylase re
action. The R-structural state of the enzyme-PALA complex is an excellent r
epresentation of the form of the enzyme that occurs at the moment in the ca
talytic cycle when the tetrahedral intermediate is formed. Finally, improve
d electron density in the N-terminal region of the regulatory chain (residu
es 1 to 7) has allowed tracing of the entire regulatory chain. The N-termin
al segments of the R1 and R6 chains are located in close proximity to each
other and to the regulatory site. This portion of the molecule may be invol
ved in the observed asymmetry between the regulatory binding sites as well
as in the hetero-tropic response of the enzyme. Protein 1999;37: 729-742, (
C) 1999 Wiley-Liss, Inc.