Comparison of aspartate transcarbamoylases from wheat germ and Escherichia coli: functionally identical histidines in nonhomologous local sequences

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Abstract
Aspartate transcarbamoylase (ATCase) from wheat germ and the catalytic subunit of the enzyme from Escherichia coli are trimers of similar size. The former is a regulatory enzyme in its trimeric state, while the latter is a component of a complex regulatory dodecamer. In a comparison of the two enzymes, reaction with the diethyl pyrocarbonate revealed a highly active, essential histidine residue in each case. The two histidines (i.e., one in each enzyme) behaved nearly identically with repsect to the following functional properties: (1) kinetics of acylation (ethoxyformylation) and concomitant inactivation; (2) kinetics of deacylation by hydroxylamine and concomitant reactivation; (3) hyperbolic dependence of the apparent first-order rate constant (kapp) on diethyl pyrocarbonate concentration; (4) pH dependence of kapp (5) failure of active-center ligands to protect the residue against diethyl pyrocarbonate, producing instead near-identical increase in the activation rate. These similarities point to an essential, highly conserved histidine in each enzyme, in a functional microenvironment that has changed relatively little since the divergence of plants and bacteria. Ethoxyformylated peptides were isolated from tryptic digests of the two inactivated enzymes. Sequencing of the major labeled peptide in each case showed the wheat and E. coli histidines embedded in nonhomologous primary segments, suggesting that, contrary to expectation, these segments are not part of the conserved microenvironment. In the case of the E. coli enzyme, the essential residue was identified as His-134 in the known sequence, which has a potential catalytic role on crystallographic evidence [Krause, K. L., Volz, K. W., and Lipscomb, W. N. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1643-1647]. A second, much less reactive histidine was identified as His-64. Since the fully primary and tertiary structures of the wheat-germ enzyme are not known, it is not possible at present to compare the environments of the essential histidine in the two enzymes.