Aspartate Transcarbamylase

The binding of small molecules to macromolecules can be studied by nuclear magnetic resonance since the width of a resonance can often be related to rotational correlation times and exchange rates for bound and unbound species. Line widths for carbon-linked protons of analogues of carbamyl phosphate (carbamyl-P) in the presence of the catalytic subunit of aspartate transcarbamylase are a measure of the rotational freedom of the bound analogues. The protons of phosphonacetamide are broadened considerably in the presence of the subunit, while those of N-methyl phosphonacetamide and methyl phosphonate are not, indicating that an interaction between the [see PDF for structure] group of the analogue and the enzyme limits rotation about the P—C bond in the first instance but not in the latter two. The resonance line width for the methylene protons of 0.025 m succinate, a competitive inhibitor of aspartate, is 0.48 Hz in the absence of enzyme at pH 7, increases slightly when catalytic subunit (20 mg per ml) is added, but increases to 2.00 Hz upon the further addition of 0.025 m carbamyl-P. In this case, the broadening is due primarily to an increase in the lifetime of the succinate-enzyme complex, induced by carbamyl-P. Some analogues of carbamyl-P, for example phosphonacetamide and acetyl phosphate, induce broadening but others, including N-methyl phosphonacetamide, N-methyl carbamyl phosphate, and methyl phosphonate, do not. Only analogues which are not larger than carbamyl-P and which have a carbonyl group in addition to a phosphate or phosphonate dianion induce the broadening of the succinate line. The resonance for the protons of malonate, another inhibitor of aspartate, is also broadened by carbamyl-P and phosphonacetamide in the presence of the catalytic subunit. Dissociation constants determined at pH 7 confirm that carbamyl-P is the most effective analogue in inducing succinate binding at this pH. In addition to line widths, chemical shifts of the bound species have been determined for some of the analogues. The chemical shifts suggest that an aromatic ring is near the binding site for phosphate.