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G C Roberts

Publications and source records attributed to G C Roberts.

At least 163 records · Page 9Linked to original sources
PubMed1981

Effects of coenzyme binding on histidine residues of Lactobacillus casei dihydrofolate reductase.

The effects of coenzyme binding on the seven histidine C2 proton resonances of Lactobacillus casei dihydrofolate reductase have been determined. Binary complexes containing NADP+, NADPH, and their hypoxanthine, thionicotinamide, and acetylpyridine analogues, together with ternary complexes containing the inhibitors trimethoprim or methotrexate, have been examined. Four of the histidine residues are affected by coenzyme binding. The largest effect-a marked upfield shift (0.85 ppm) of the C2 proton resonance-is seen for His-64. The hypoxanthine analogue of the coenzyme was found to produce a smaller upfield shift and, in addition, a decrease in the pK of His-64. The effects on this reductase are discussed in the light of the crystal structure [Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J. Biol. Chem. 253, 6946], and it is concluded that His-64 is close to a carboxyl group in the free enzyme and that the hypoxanthine ring binds in a somewhat different orientation to the adenine ring. The effects on histidine resonances A, E, and G are significantly different for oxidized and reduced coenzymes. The changes in pK of the histidines giving rise to resonances A and E (probably His-22 and His-18) are discussed in terms of ligand-induced conformational changes, which differ for NADP+ and NADPH.

Binding Sites↗
PubMed1981

Hydrogen-1 nuclear magnetic resonance study of the complexes of two diastereoisomers of folinic acid with dihydrofolate reductase.

The 1H chemical shifts for the formyl and benzoyl protons of the individual diastereoisomeres of folinic acid bound to dihydrofolate reductase have been measured. For the tightly bound biologically active 6S, alpha S isomer, the "bound" signals were assigned by using transfer of saturation methods. In this case, only one of the two rotameric states of the formyl group in folinic acid (form I) is bound to the enzyme. The H3' and H5' benzoyl protons have identical shifts in the bound state (as do the H2' and H6' protons). This equivalence is attributed to flipping of the benzoyl ring about the N10-C4' and C1'-CO bonds in the bound state. In the case of the biologically inactive 6R, alpha S isomer, both rotameric forms (I and II) bind to the enzyme. The "bound" shifts for the formyl and aromatic protons are different in the complexes with the 6S, alpha S and 6R, alpha S isomers, indicating that the pteridine ring and benzoyl moiety are binding in different environments in their enzyme complexes. The glutamic acid moiety is probably binding at the same site in the two complexes.

Lacticaseibacillus casei↗
PubMed1980

Proton nuclear magnetic resonance study of the self-complementary hexanucleotide d(pTpA)3 and its interaction with daunomycin.

The helix-coil transition of the self-complementary hexanucleotide d(pTpA)3 has been studied in 1 M NaCl by high-resolution proton nuclear magnetic resonance spectroscopy. Almost all of the 12 resonances deriving from the three environments of the four nucleotide protons have been assigned to the central, internal, or terminal nucleotides. At 5 degrees C, the effect of extensive fraying is evident since the central base pairs exhibit of extensive fraying is evident since the central base pairs exhibit only 20% of the chemical shifts observed for poly(dA-dT) . poly(dA-dT) accompanying denaturation. Daunomycin interacts with the hexanucleotide duplex at 5 degrees C and stabilizes it by 21 degrees C at a drug/nucleotide ratio of 0.063 (i.e., drug/hexanucleotide duplex ratio of 0.75). The chemical shifts of the drug protons suggest that ring D of daunomycin does not overlap significantly with the central base pairs of the hexanucleotide and that it extends out from the "helix". This information, together with studies of space-filling models of the complex, suggests that rings B and C of daunomycin overlap with adjacent base pairs and are skewed with respect to the base pairs.

Daunorubicin↗
PubMed1980

Binding of coenzyme analogues to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes.

The binding, or association, constants of NADP+ NADPH, and a series of structural analogues to dihydrofolate reductase from Lactobacillus casei MTX/R have been determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogues of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrxate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this "cooperativity", which covers a range of 1-37 fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analogue; the nicotinamide ring of the coenzymes is clearly important in these effects. The binding constants of the reduced coenzymes in the presence of methotrexate or trimethoprim were too high to be measured fluorometrically. The dissociation rate constants of the coenzymes from their ternary complexes were therefore measured and compared with the values for the binary complexes reported by Dunn and co-workers [Dunn, S. M. J., Bathchelor, J. G., & King, R. W.(1978) Biochemistry 17, 2356]. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant--by factors of 300-2200. The binding constant of methotrexate in the ternary complex is calculated to be approximately 1.3 X 10(12) M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogues are discussed in the light of information from crystallography and NMR spectroscopy.

Kinetics↗
PubMed1980

Effects of coenzyme analogues on the binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to Lactobacillus casei dihydrofolate reductase.

The binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to dehydrofolate reductase from Lactobacillus casei MTX/R in the presence of a series of co-enzymes and coenzyme analogues has been measured fluorometrically. These two ligands, which can be regarded as "fragments" of the powerful inhibitor methotrexate, have been shown to bind cooperatively in the absence of coenzyme [Birdsall, B., Burgen, A. S. V., Rodrigues de Miranda, J., & Roberts, G. C. K. (1978) Biochemistry 17, 2102], p-amino-benzoyl-L-glutamate binding 58 times more tightly in the presence of 2,4-diaminopyrimidine than in its absence. In the presence of coenzymes, this cooperativity ranges from 1.8- to 428-fold. The effects of coenzymes on individual binding steps range from an 8-fold decrease in binding constant to a 23-fold increase. The structural specificity of these effects are discussed in terms of a model involving ligand-induced conformational changes and compared with the effects on trimethoprim and methotrexate binding described in the preceding paper [Birdsall, B., Burgen, A. S. V., & Roberts, G. C. K. (1980) Biochemistry (first paper of four in this issue)].

4-Aminobenzoic Acid↗
PubMed1980

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase.

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.

Binding Sites↗
PubMed1980

Phosphorus-31 nuclear magnetic resonance studies of the binding of oxidized coenzymes to Lactobacillus casei dihydrofolate reductase.

The 31P NMR spectra of NADP+ and a number of its structural analogues have been obtained from their binary and ternary complexes with Lactobacillus casei dihydrofolate reductase. The 2'-phosphate resonance is shifted downfield 2.7-2.9 ppm in all cases. Line-shape analysis of this resonance as a function of coenzyme concentration gave values for the dissociation rate constant of the coenzyme from many of the complexes. The values obtained are discussed in terms of the kinetic mechanism of coenzyme binding. The chemical shifts of the pyrophosphate resonances vary from one complex to another over a range of 3.8 ppm. The assignment of these signals to the individuals pyrophosphate 31P nuclei and the structural origins of the chemical shift changes are discussed. From these data, and the 1H NMR experiments describedin the preceding paper [Hyde, E. I., Birdsall, B., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1980) Biochemistry (third paper of four in this issue)], it is concluded that the "nicotinamide" end of the thionicotinamide and acetylpyridine coenzyme analogue binds to the enzyme quite differently from that of the natural coenzyme NADP+.

Binding Sites↗
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↗
PubMed1980

The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.

The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.

Bromosuccinimide↗
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↗
PubMed1979

1H nuclear-magnetic-resonance studies of porcine lutropin and its alpha and beta subunits.

The titration curves of the histidine residues of porcine lutropin and its isolated alpha and beta subunits have been determined by following the pH-dependence of the imidazole C-2 proton resonances. The isolated alpha subunit contains a buried histidine, whose C-2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2). The four histidine C-2 proton resonances have been assigned to specific residues in the amino-acid sequence, by means of deuterium and tritium exchange experiments on the alpha subunit and its des(92-96) derivative. The histidine with a pK of 3.3 is identified as His-alpha87. The effects of pH on tyrosine and methyl proton resonances show that the titration of His-87 in the isolated alpha subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition. The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.

Animals↗
PubMed1979

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.

Circular Dichroism↗