Characterization of a buried neutral histidine residue in<i>Bacillus circulans</i>xylanase: Nmr assignments, pH titration, and hydrogen exchange

Plesniak, Leigh A.; Connelly, Gregory P.; Wakarchuk, Warren W.; McIntosh, Lawrence P.

doi:10.1002/pro.5560051118

Cited by 68 publications

(96 citation statements)

References 62 publications

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“…For simplicity, these proteins will hereafter be referred to as BCX. All HX experiments and the D/H fractionation factor measurements were performed on the wild-type xylanase, while 15 N relaxation and studies of the bound water interactions were carried out using both the 13 NMR Spectroscopy. NMR spectra were recorded with a Varian Unity spectrometer operating at 500 MHz for proton and equipped with a pulsed-field gradient 1 H/ 13 C/ 15 N probe.…”

Section: Methodsmentioning

confidence: 99%

“…Exchange rates were measured in the same manner for the two tyrosine hydroxyl H η protons with chemical shifts at 11.6 and 12.7 ppm. 15 (18,20). These data were used to determine k R and k N , the rate constants for magnetization exchange between the H 2 proton of His149 and water protons during the ROE and NOE mixing periods, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Representative spectra can be found in Figure 6 of Plesniak et al (15). A plot of the log k ex versus pD has a V or chevron shape, indicative of both specific acid-and basecatalyzed exchange (Figure 2a).…”

Section: Hydrogen-deuterium Exchange Of His149 Hmentioning

confidence: 97%

“…In addition to the peak from His149 H 2 , two unusually downfield resonances are detected at 11.6 and 12.7 ppm in the 1 H NMR spectrum of BCX (15). These remain as singlets when the protein is 13 C-and 15 N-labeled and thus must arise from protons bonded to O or S atoms.…”

Section: )mentioning

confidence: 98%

“…Previously, we have characterized the two histidines in BCX using NMR spectroscopy (15). Whereas the exposed His156 has a relatively unperturbed pK a of ∼6.5, the buried His149 remains in the neutral N 2 H tautomeric form under all conditions examined.…”

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confidence: 97%

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Characterization of a Buried Neutral Histidine inBacillus circulansXylanase: Internal Dynamics and Interaction with a Bound Water Molecule

Connelly

McIntosh

1998

Biochemistry

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NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule. Rate constants for the specific acid-and base-catalyzed exchange following bimolecular kinetics (EX 2 ) of the nitrogen-bonded H 2 of this buried, neutral histidine were determined. At pD min 7.0 and 30°C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of ∼10 7 relative to that predicted for an exposed histidine. The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations. Consistent with its buried environment, the N 2 -H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S 2 ) 0.83 determined from 15 N relaxation measurements. The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H 2 of His149. Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H 2 and a water molecule with the same chemical shift as the bulk solvent. However, the chemical shift of H 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 ( 0.02) indicate that this is not a so-called low-barrier hydrogen bond. Lower and upper bounds on the lifetime of the internal water are estimated to be 10 -8 and 10 -3 s. Therefore the chemical exchange of solvent protons with those of His149 H 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude.The native structure of a protein is stabilized under physiological conditions by a delicate balance of entropic, hydrophobic, electrostatic, hydrogen-bonding, and van der Waals interactions. On an individual basis these interactions are comparable to thermal energies, and thus transient structural fluctuations, up to and including global unfolding, are constantly occurring in any ensemble of protein molecules. Understanding the contributions of this inherent flexibility toward the structure, stability, and functions of these molecules remains an important experimental and theoretical challenge. The dynamic behavior of proteins is amenable to study by a wide array of techniques, including nuclear magnetic resonance (NMR) 1 spectroscopy and hydrogen exchange (HX). While HX studies have typically focused on the slowly exchanging main-chain amides in proteins, other labile groups can also provide structural and dynamic information. The side chains of histidine residues are one such example. Normally the nitrogen-bonded H δ1 and H 2 of the imidazole ring exchange with the solvent too rapidly to be detected by standard NMR methods. However, when their exchange is slowed by burial within the core of a protein and/or by hydrogen bonding, measurement of their proton exchange rates can provide useful informatio...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Hydrogen-deuterium Exchange Of His149 Hmentioning

confidence: 97%

Section: )mentioning

confidence: 98%

mentioning

confidence: 97%

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Characterization of a Buried Neutral Histidine inBacillus circulansXylanase: Internal Dynamics and Interaction with a Bound Water Molecule

Connelly

McIntosh

1998

Biochemistry

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Exploring protein interiors: The role of a buried histidine in the KH module fold

Fraternali¹,

Amodeo²,

Musco³

et al. 1999

Proteins

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Identification of a Catalytic Base for Sugar Oxidation in the Pyranose 2‐Oxidase Reaction

2011

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Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK(a) >10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK(a) value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K(d) for D-glucose binding was 19 times greater.

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Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange

Cited by 68 publications

References 62 publications

Characterization of a Buried Neutral Histidine inBacillus circulansXylanase: Internal Dynamics and Interaction with a Bound Water Molecule

Characterization of a Buried Neutral Histidine inBacillus circulansXylanase: Internal Dynamics and Interaction with a Bound Water Molecule

Exploring protein interiors: The role of a buried histidine in the KH module fold

Identification of a Catalytic Base for Sugar Oxidation in the Pyranose 2‐Oxidase Reaction

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