Ligand-induced isomerizations of Escherichia coli ornithine transcarbamoylase. An ultraviolet difference analysis.
Ligand-induced ultraviolet difference spectra have been determined for Escherichia coli ornithine transcarbamoylase. The most prominent feature of the spectra is an absorbance difference which resembles a single period of a sine wave spanning the 245-320 nm region with a maximum at approximately 270 nm and a minimum at around 295-300 nm. This broad absorbance difference is typical of a blue-shift 1La band of tryptophan. Superimposed on the broad band in the 275-310 nm region is a series of smaller, narrow peaks resulted from red-shifted 1Lb bands of tryptophan and tyrosine residues. At pH 8.5, only carbamoyl phosphate and its analog phosphonacetamide yield a large ultraviolet difference absorbance (approximately 1800 M-1 cm-1) when bound to the enzyme. The spectra obtained are essentially the same in lineshape to and 80% in intensity of that produced by the bisubstrate analogy, N-(phosphonacetyl)-L-ornithine. In contrast, inorganic phosphate, a product of the reaction, induces small protein absorbance changes (approximately 300 M-1 cm-1) mainly in the 275-310 nm range. When complexed to the free enzyme, L-ornithine yields a marginally discernible ultraviolet difference spectrum in the 275-310 nm region, and its analogs L-norvaline and L-citrulline provide no absorbance change. However, inorganic phosphate in combination with any of the L-amino acids produces a difference spectrum similar to that given by carbamoyl phosphate alone. Collectively, these spectra suggest that carbamoyl phosphate elicits an isomerization required for the formation of the ternary complex and are consistent with the compulsory ordered mechanism of the enzyme at pH 8.5 with carbamoyl phosphate being the first substrate bound. Below pH 8, there is a kinetically discernible amount of random binding, but ordered addition is still the preferred pathway (Wargnies B., Legrain, C., and Stalon, V. (1978) Eur J. Biochem. 89, 203-212). Reflecting this change, the difference absorbance of the enzyme bound with carbamoyl phosphate is also pH dependent. The 1La band in the carbamoyl phosphate difference spectrum diminishes by approximately 20% at low pH. The PALO-induced changes, however, are pH invariant suggesting that full extent of the induced-fit isomerization is always reached in the ternary complex.
Department of Chemistry, Metcalf Center for Science and Engineering, Boston University, Massachusetts 02215.
A W
AW
Miller
L C
LC
Kuo
D002221:Carbamyl Phosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007536:Isomerism; D008024:Ligands; D009952:Ornithine / Q000031:analogs & derivatives / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014633:Valine / Q000378:metabolism / Q000031:analogs & derivatives
D008024:Ligands; D002221:Carbamyl Phosphate; C020464:N(delta)-(phosphonoacetyl)-L-ornithine; C005313:norvaline; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D014633:Valine