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J Feeney

Publications and source records attributed to J Feeney.

At least 145 records · Page 8Linked to original sources
PubMed1980

Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei.

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.

Bromosuccinimide↗
PubMed1980

Photo-CIDNP studies of the influence of ligand binding on the surface accessibility of aromatic residues in dihydrofolate reductase.

The surface accessibility of the histidine, tyrosine, and tryptophan residues of Lactobacillus casei dihydrofolate reductase has been determined from 360-MHz 1H photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR experiments. In the absence of ligands, four (or perhaps five) of the seven histidine residues and at least one of the four tryptophan residues are accessible to a flavin dye molecule. One of the five tyrosine residues is also slightly accessible. Of the accessible histidine residues, one becomes inaccessible on the binding of NADP+ and one on the binding of p-aminobenzoyl glutamate. These have been assigned to residues which interact directly with these two ligands. One histidine residue (probably His-22) shows an increase in accessibility on addition of folate or methotrexate to the enzyme . NADP+ complex. In addition, the binding of several ligands, notably trimethoprim, leads to an increase in the accessibility of a tryptophan residue. This is clear evidence for ligand-induced conformational changes in dihydrofolate reductase and allows us to identify some of the residues involved.

Folic Acid↗
PubMed1979

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.

Binding Sites↗
PubMed1975

A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase.

The binding of NADP+ to dihydrofolate reductase (EC 1.5.1.3) in the presence and absence of substrate analogs has been studied using 1H and 13C nuclear magnetic resonance (NMR). NADP+ binds strongly to the enzyme alone and in the presence of folate, aminopterin, and methotrexate with a stoichiometry of 1 mol of NADP+/mol of enzyme. In the 13C spectra of the binary and ternary complexes, separate signals were observed for the carboxamide carbon of free and bound [13CO]NADP+ (enriched 90% in 13C). The 13C signal of the NADP+-reductase complex is much broader than that in the ternary complex with methotrexate because of exchange line broadening on the binary complex signal. From the difference in line widths (17.5 +/- 3.0 Hz) an estimate of the dissociation rate constant of the binary complex has been obtained (55 +/- 10 sec-1). The dissociation rate of the NADP+-reductase complex is not the rate-limiting step in the overall reaction. In the various complexes studied large 13C chemical shifts were measured for bound [13CO]NADP+ relative to free NADP+ (upfield shifts of 1.6-4.3 ppm). The most likely origin of the bound shifts lies in the effects on the shieldings of electric fields from nearby charged groups. For the NADP+-reductase-folate system two 13C signals from bound NADP+ are observed indicating the presence of more than one form of the ternary complex. The IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments. Substantial changes in the IH spectrum of the protein were observed in the different complexes and these are distinct from those seen in the presence of NADPH.

Amides↗