Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

Poe, Martin; Hoogsteen, Karst; Matthews, D.A.

doi:10.1016/s0021-9258(19)86867-9

Cited by 15 publications

(5 citation statements)

References 38 publications

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“…The titration of H2' can be fit to a pKa of 8.6 and may possibly be associated with the same histidine residue as resonance H2. pAfa values of 8.0 and higher for histidyl residues in proteins have been reported in the literature (Stoesz et al, 1979;Arata et al, 1980;Poe et al, 1979; Markley, 1975b) and may be explained in the case of ABP by the involvement of the histidine residues in ion-pair interactions with nearby acidic residues as previously identified in crystallographic studies of ABP (Guilliland & Quiocho, 1981).…”

Section: Resultssupporting

confidence: 52%

“…The pXa values of the histidine residues that we observe (pXa values of 8.0 and 8.6) are higher than normal (pXa 6.9), presumably due to their ionic interactions with carboxyl groups. A histidine residue involved in an ion-pair interaction in another protein has been reported to have a pXa value greater than 10 (Stoesz et al, 1979), and many others have been reported with pKa values near pH 8 (Arata et al, 1980;Poe et al, 1979;Markley, 1975b). In our studies, one histidine resonance, H2, has been shown to be sensitive to the binding of L-arabinose.…”

Section: Discussionsupporting

confidence: 56%

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Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein

Clark¹,

Gerken²,

Hogg³

1982

Biochemistry

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The L-arabinose binding protein (ABP) from Escherichia coli was studied by proton nuclear magnetic resonance spectroscopy (1H NMR). Distinct spectral changes occur when ABP binds its natural ligand, L-arabinose, which involve resonances in the aromatic ring current shifted methyl, bulk methyl, methylene, aromatic, and amide proton regions of the spectra. Several amide resonances can be "protected" from deuterium exchange if L-arabinose is bound to ABP prior to deuterium oxide dialysis. On the basis of the pH dependence of their chemical shifts, two low-field resonances have been tentatively assigned to C2 protons of two of the three histidines present in ABP. These histidyl residues have pK values of 8.0 and 8.6 which support their involvement in ionic interactions observed earlier in the crystallographic analysis. One histidyl residue shows a small chemical shift change upon the addition of arabinose. When ABP binds D-galactose, changes in the spectra occur which are different than those observed when L-arabinose is bound. Binding of L-arabinose and D-galactose to the binding protein (ABP) was considered by equilibrium binding and fluorescence emission spectroscopy. ABP binds L-arabinose and D-galactose with high affinities (Kd's at 6 degrees C of 1.3 x 10(-7) and 1.9 x 10(-7) M, respectively), and both enthalpy and entropy contribute to the ABP-ligand association. When excited at 285 nm, ABP has a fluorescence emission maximum of 340 nm which is quenched and blue shifted (to 337 nm) upon binding L-arabinose. ABP binding D-galactose produced a similar emission shift but no fluorescence quenching.

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Section: Resultssupporting

confidence: 52%

Section: Discussionsupporting

confidence: 56%

Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein

Clark¹,

Gerken²,

Hogg³

1982

Biochemistry

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“…¿His-141 shows a moderately high pK (7.2-7.4). Normal His pA values in this enzyme are about 6 determined by NMR; T = 25 eC (Poe et al, 1979). 'pH difference titration of native and the 0-ethyl ester derivative of Asp-52 results in a change of the apparent pA of Glu-35 by 1.1 units.…”

Section: Applicationsmentioning

confidence: 87%

Electrostatic effects in water-accessible regions of proteins

Mehler¹,

Eichele²

1984

Biochemistry

135

115

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“…Histidine G has been assigned to histidine-153 by Matthews (1979), and Wyeth et al (1980) feel that it could be either histidine-153 or histidine-89. However, histidine-153 is homologous with histidine-149 in dihydrofolate reductase from E. coli (Freisheim et al, 1978), which reacts rapidly with diethyl pyrocarbonate (Poe et al, 1979). Poe et al (1979) found that histidine-124 of dihydrofolate reductase from E. coli reacted poorly, if at all, but the homologous region of the enzyme from L. casei has no histidine residues.…”

Section: Discussionmentioning

confidence: 99%

“…However, histidine-153 is homologous with histidine-149 in dihydrofolate reductase from E. coli (Freisheim et al, 1978), which reacts rapidly with diethyl pyrocarbonate (Poe et al, 1979). Poe et al (1979) found that histidine-124 of dihydrofolate reductase from E. coli reacted poorly, if at all, but the homologous region of the enzyme from L. casei has no histidine residues. Another possible interpretation is that all histidine residues are potentially reactive and the unreacted residues are randomly selected and are distributed among all, or a restricted group, of the total histidine residues.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate

Daron¹,

Aull²

1982

Biochemistry

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The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.

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Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

Cited by 15 publications

References 38 publications

Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein

Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein

Electrostatic effects in water-accessible regions of proteins

Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate

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