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J. Biol. Chem., Vol. 258, Issue 6, 3528-3538, 03, 1983

Communication between catalytic and regulatory subunits in Ni(II)- and Co(II)-aspartate transcarbamoylase. Ligand-promoted structural alterations at the intersubunit bonding domains

RS Johnson and HK Schachman

The cooperativity with respect to substrates exhibited by allosteric enzymes as well as their inhibition and activation by effectors is mediated through the propagation of conformational changes from the site of ligand binding on one polypeptide chain to other, unliganded chains. This "communication" between subunits in the enzyme is achieved in part by changes at the intersubunit bonding domains as the oligomeric enzymes are converted from the low affinity state to a conformation of high affinity. Structural alterations at the bonding domains between the catalytic and regulatory subunits of aspartate transcarbamoylase from Escherichia coli were detected in derivatives containing either nickel or cobalt in place of the endogenous zinc ion bound to each of the six regulatory chains. These metal-substituted derivatives exhibited physical-chemical and allosteric properties identical to those of the native enzyme. Spectral analyses indicated that both nickel and cobalt are bound to the enzyme in a distorted tetrahedral environment. With the nickel ions serving as spectral probes for structural alterations at the intersubunit bonding domains, it was observed that the substrate, carbamoyl phosphate, and the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate caused different changes. The substrate analog, succinate, alone had no effect. However, the combination of both carbamoyl phosphate and succinate produced the same spectral change as that caused by the bisubstrate analog. Circular dichroism measurements on the nickel derivative showed that the perturbation at 360 nm was directly proportional to the extent of occupancy of the six active sites by the bisubstrate ligand. In contrast, the changes at 406 and 460 nm coincided with the gross conformational change measured by the decrease in the sedimentation coefficient of the enzyme. These latter changes in the circular dichroism and the alterations in the absorption spectrum were complete even though about one-third of the active sites were unliganded.
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