Alkaline phosphatase (EC 3.1.3.1) containing m-fluorotyrosine has been prepared from E. coli grown in the presence of m-fluorotyrosine. The kinetic properties of the m-fluorotyrosine enzyme measured with p-nitrophenylphosphate at pH 8.0 and dinitrophenylphosphate at pH 5.5 are essentially the same as those of normal alkaline phosphatase. However, the ability of the m-fluorotyrosine protein to refold active enzyme after acid denaturation, while unchanged at pH 5.8, was markedly decreased at pH 7.6. This result implies that the tyrosines must be in their protonated form for the protein to refold, reassociate, and take on zinc. intensity, and 1H chemical shifts, coupling constants, and relaxation times are the best understood. However, 1H chemical shifts are confined to a narrow 10-ppm range so that even at high frequencies (100-300 MHz), the 1H NMR spectra of all but the smaller proteins are unresolved envelopes.Several approaches have been used to simplify protein 1H NMR spectra, including specific proton insertion of the protein (2), difference NMR methods (3), and the use of NMR "shift reagents" (4). The protein that we have chosen to study is the alkaline phosphatase (EC 3.1.3.1.) produced by Escherichia coli. This enzyme is a zinc metalloprotein consisting of two identical subunits, and an extensive body of information has accumulated over the past 10 years concerning its structure and function (see ref. 8). It offers the additional advantage that the bacterial system can be manipulated to allow for almost exclusive incorporation of a fluorine-containing amino acid into protein and for the production of enzyme primarily during that portion of the cells' growth when the analogue is present. In previous studies with E. coli alkaline phosphatase, ana: logues of arginine (canavanine), histidine (2-methylhistidine and triazolealanine), proline (azetidine-2-carboxylate), phenylalanine (fluorophenylalanine), and tryptophan (azatryptophan and tryptazan) have been inserted into the enzyme with varying results. Only analogues of the latter two natural amino acids allowed active enzyme to be formed (9, 10). All others produced aberrant, inactive subunits (10). In the current study, we prepared enzyme from bacteria grown in the presence of fluorotyrosine. We wished to determine whether the fluorotyrosine residues in a protein as large as E. coli alkaline phosphatase (molecular weight = 86,000) would be useful as NMR probes for analyzing conformational changes in proteins.
MATERIALS AND METHODSFluorotyrosine alkaline phosphatase was isolated from a tyrosine auxotroph of E. coli W3747 (American Type Culture Collection No. 27256) (11). Cells were transferred from agar plates containing a Tris-buffered minimal salts medium (12) supplemented with L-tyrosine (20 ug/ml) and L-methionine (20 ug/ml), 0.2% glucose, and Pi (6 X 10-4 M) to flasks containing the same media, except that the tyrosine and Pi were replaced with pepticase (Sheffield Scientific Co., at 5 mg/ml). This amount of pepticase provided a level of Pi equal...
