DOMAIN CLOSURE IN THE CATALYTIC CHAINS OF ESCHERICHIA-COLI ASPARTATE TRANSCARBAMOYLASE INFLUENCES THE KINETIC MECHANISM

The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T --> R transition, is stabilized by salt-bridges between Glu-50 a nd both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzy me toward a low activity, low affinity state (Newton, C. J., and Kantr owitz, E. R. (1990) Biochemistry, 29, 1444-1451), Kinetic isotope effe cts (KIE) and equilibrium isotope exchange kinetics (EIEK) have been u sed to probe the dynamic properties of the Glu-50 --> Ala enzyme. Unli ke the behavior of the wild-type enzyme, the observed kinetic isotope effect for C-13 versus C-12 at the carbonyl group of carbamoyl phospha te (CP) increased upon the binding of ligands which promote the format ion of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [C-14]Asp reversible arrow Carbamoyl asparta te (CAsp) exchange with the Glu-50 --> Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [C-14]Cp reve rsible arrow CAsp exchange was only 50-fold slower, reversing the rela tive rates observed with the wild-type enzyme. In addition, upon varia tion of substrate pairs involving Asp or CAsp, loss of inhibition effe cts in the CP reversible arrow CAsp exchange indicated that the Glu-50 --> Ala substitution caused the kinetic mechanism for the mutant enzy me to shift from ordered to random. Computer simulations of the EIEK d ata indicate that the Glu-50 --> Ala mutation specifically causes stro ng decreases in the rates of catalysis and association-dissociation fo r Asp and CAsp, with minimal effects on the GP and P-i on-off rates, W ith substrates bound, the Glu-50 --> Ala enzyme apparently does not at tain a full R-state conformation. The PALA-activated Glu-50 --> Ala en zyme, however, exhibits substrate affinities comparable to those for t he wild-type enzyme, but fails to restore the preferred order substrat e binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50 --> Ala enzyme contribute to catalysis. A third state, I, is p roposed for the Glu-50 --> Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantl y to overall rate limitation.