Aspartate Transcarbamylase: Studies of the catalytic subunit by ultraviolet difference spectroscopy

Abstract

Abstract The binding of substrates and substrate analogues to the catalytic subunit of aspartate transcarbamylase from Escherichia coli can be detected by difference spectroscopy between 275 and 310 mµ; the chromophores giving rise to these difference spectra are tentatively identified as a tryptophyl residue perturbed by the binding of carbamyl phosphate and a tyrosyl residue perturbed by the binding of l-aspartate. Dissociation constants for several l-aspartate analogues have been determined by difference spectroscopy. When the carbamyl phosphate site is unoccupied, l-aspartate, succinate, l-malate, and d-malate each bind weakly at the l-aspartate site. In the presence of carbamyl phosphate, only succinate and d-malate, the two dicarboxylic acids which lack l-α-substituents, bind tightly to the enzyme. In striking contrast, when carbamyl phosphate is replaced by phosphate, only l-aspartate and l-malate, the two dicarboxylic acids which have l-α-substituents, bind tightly to the enzyme. The magnitudes of the difference spectra for the ternary complexes are substantially greater than the sum of the magnitudes of the binary complexes formed by each analogue individually only when binding of the dicarboxylic acid is enhanced. We suggest, as an explanation for these observations, that the enzyme converts a portion of the electrostatic binding energy of l-aspartate into energy for the mechanical act of forcing the substrates together. A detailed model for the catalytic mechanism is presented, based on the data presented in this and the accompanying papers.