NMR localization of protons in critical enzyme hydrogen bonds.
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ABSTRACT: Using liquid-state NMR spectroscopy we have estimated the proton-donating ability of Zn-bound water in organometallic complexes designed as models for the active site of the metalloenzyme carbonic anhydrase (CA). This ability is important for the understanding of the enzyme reaction mechanism. The desired information was obtained by (1)H and (15)N NMR at 180 K of solutions of [Tp(Ph,Me)ZnOH] [1, Tp(Ph,Me) = tris(2-methyl-4-phenylpyrazolyl)hydroborate] in CD(2)Cl(2), in the absence and presence of the proton donors (C(6)F(5))(3)BOH(2) [aquatris(pentafluorophenyl)boron] and Col-H(+) (2,4,6-trimethylpyridine-H(+)). Col-H(+) forms a strong OHN hydrogen bond with 1, where the proton is located closer to nitrogen than to oxygen. (C(6)F(5))(3)BOH(2), which exhibits a pK(a) value of 1 in water, also forms a strong hydrogen bond with 1, where the proton is shifted slightly across the hydrogen-bond center toward the Zn-bound oxygen. Finally, a complex between Col and (C(6)F(5))(3)BOH(2) was identified, exhibiting a zwitterionic OHN hydrogen bond, where H is entirely shifted to nitrogen. The comparison with complexes of Col with carboxylic acids studied previously suggests that, surprisingly, the Zn-bound water exhibits in an aprotic environment a similar proton-donating ability as a carboxylic acid characterized in water by a pK(a) of 2.2 ± 0.6. This value is much smaller than the value of 9 found for [Zn(OH(2))(6)](2+) in water and those between 5 and 8 reported for different forms of CA. Implications for the biological function of CA are discussed.Journal of the American Chemical Society 06/2011; 133(29):11331-8. · 10.68 Impact Factor
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ABSTRACT: Intramolecular and intermolecular hydrogen bonding in electronic excited states of calixarene building blocks bis(2-hydroxyphenyl)methane (2HDPM) monomer and hydrogen-bonded 2HDPM-H(2)O complex were studied theoretically using the time-dependent density functional theory (TDDFT). Twenty-four stable conformations (12 pairs of enantiomers) of 2HDPM monomer have been found in the ground state. From the calculation results, the conformations 1a and 1b which both have an intramolecular hydrogen bond are the most stable ones. The infrared spectra of 2HDPM monomer and 2HDPM-H(2)O complex in ground state and S(1) state were calculated. The stretching vibrational absorption band of O(2) - H(3) group in the monomer and complex disappeared in the S(1) state. At the same time, a new strong absorption band appeared at the C=O stretching region. From the calculation of bond lengths, it indicates that the O(2) - H(3) bond is significantly lengthened in the S(1) state. However, the C(1) - O(2) bond is drastically shortened upon electronic excitation to the S(1) state and has the characteristics of C=O band. Furthermore, the intramolecular hydrogen bond O(2) - H(3) · · · O(4) of the 2HDPM monomer and the intermolecular hydrogen bonds O(2) - H(3) · · · O(7) and O(7) - H(9) · · · O(4) of 2HDPM-H(2)O complex are all shortened and strengthened in the S(1) state.Journal of Molecular Modeling 01/2013; · 1.98 Impact Factor
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ABSTRACT: Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.Journal of the American Chemical Society 08/2011; 133(37):14823-30. · 10.68 Impact Factor
NMR Localization of Protons in Critical Enzyme Hydrogen Bonds
Shasad Sharif,‡Emily Fogle,†Michael D. Toney,†Gleb S. Denisov,§Ilya G. Shenderovich,‡
Gerd Buntkowsky,¶Peter M. Tolstoy,‡Monique Chan Huot,‡and Hans-Heinrich Limbach*,‡
Institut fu ¨r Chemie und Biochemie, Takustrasse 3, Freie UniVersita ¨t Berlin, D-14195 Berlin, Germany, Institute of
Physics, St. Petersburg State UniVersity, 198504 St. Petersburg, Russian Federation, Department of Chemistry,
UniVersity of CaliforniasDaVis, DaVis, California 95616, and Institut fu ¨r Physikalische Chemie, Friedrich Schiller
UniVersity Jena, Helmholtzweg 4, D-07743 Jena, Germany
Received April 23, 2007; E-mail: firstname.lastname@example.org
Knowledge of proton positions in mechanistically critical H-
bonds in enzyme active sites is essential for understanding their
great catalytic powers. Unfortunately, it is difficult to localize
protons using X-ray diffraction. Neutron diffraction has been rarely
applied and only to proteins below ∼30 kDa.1The main drawback
of using neutron diffraction in protein structure determination has
been the requirement for relatively large protein single crystals.2
NMR methods exploiting dipolar couplings are also limited to
smaller biopolymers.3In this work, we demonstrate that a mecha-
nistically critical proton in a large enzyme can be localized by
combining NMR studies of enzyme and model systems, a stratagem
that does not require single crystals.
We studied E. coli aspartate aminotransferase (AspAT, ∼88
kDa), a pyridoxal-5′-phosphate (PLP, vitamin B6)-dependent en-
zyme that catalyzes reactions of various amino acids.4The PLP
cofactor is covalently bound to LYS258 as an “internal” aldimine
in the active site of AspAT (Scheme 1).5Its pyridine nitrogen forms
an OHN hydrogen bond to the side-chain carboxylate oxygen of
ASP222. An O‚‚‚N distance of 2.64 Å was determined, but the
proton was not localized.5b
It has been postulated that the proton must be located on the
pyridine nitrogen to give a zwitterionic structure of the intramo-
lecular OHN hydrogen bond between the phenolic O and the
aldimine N (Scheme 1) to enable AspAT catalytic activity.4Indeed,
removal of the carboxylate group from the position 222 side chain
by the D222A mutation indeed produces an inactive enzyme.6
Recent X-ray diffraction as well as solid and liquid state NMR
studies on model systems have confirmed this cooperative H-bond
coupling,7which is suppressed in aqueous solution.7cMoreover, it
was shown for pyridine rings in aldimine model systems7,8by
dipolar NMR that their15N chemical shifts can be used to estimate
the OHN hydrogen bond geometries, which in one case was in
excellent agreement with neutron diffraction.7bHere, we extend
this method to estimate the geometry of the critical intermolecular
OHN hydrogen bond in the active site of AspAT and show that
acid-base behavior in enzymes is better modeled using aprotic
polar rather than aqueous solutions.
Figure 1a depicts the solid state15N CPMAS NMR spectrum of
microcrystalline holo-AspAT containing15N in the pyridine ring
of PLP synthesized as described previously.7bIts
resonates at 175 ppm, whereas the signals of the nitrogen atoms of
the AspAT backbone appear around 82 ppm (confirmed by removal
of PLP-15N). Using the15N chemical shift distance correlation for
PLP models,7we find an estimated N-H distance of 1.09 Å and a
H‚‚‚O distance of 1.54 Å. This clearly indicates a zwitterionic
structure for the intermolecular OHN hydrogen bond in AspAT.
The estimated errors are given in Table 1 and contain a contribution
from the signal width and another from the neglect of the difference
between the mean average distances and the inverse cubic distances
measured by dipolar NMR. The sum of the OH and HN distance
estimated is in excellent agreement with the crystallographic O‚‚
‚N distance of 2.64 Å.5bThe distance was reported to shorten to
2.58 Å when maleate (an inhibitor) binds to AspAT, leading to the
closed enzyme conformation.5bThis result is confirmed here by
NMR (Table 1).
The15N signal of holo-AspAT in aqueous solution at pH 7.5
(Figure 1c) exhibits a small shift to 167 ppm, consistent with a
slight shortening of the N‚‚‚H distance and a slight increase of the
H‚‚‚O distance. Thus, the pyridine nitrogen remains protonated
under physiological conditions. However, the pKaof the pyridine
N of the methylamine-PLP aldimine in water is 5.8;7cthat is, this
molecule is deprotonated at pH 7.6, leading to a signal at 262 ppm
(Figure 1d). This indicates a weak hydrogen bond to surrounding
water molecules, with an estimated H‚‚‚N distance of 1.73 Å.
Comparison with Figure 1c begs the following question: What
properties of the AspAT actiVe site enforce protonation of the
pyridine N of PLP?
The answer comes from model studies of aldimines in polar
aprotic solvents. Figure 1e depicts the15N spectrum of a model
aldimine in the freon mixture8dCDF3/CDClF2. The chemical shift
of 277 ppm is typical for the free base. The formation of a 1:1
complex with the ASP222 model Boc-Asp-OtBu gives a signal at
238 ppm (Figure 1f), corresponding to a H‚‚‚N distance of 1.43 Å.
The 2:1 complex (Figure 1f) resonates at 181 ppm, characterized
by a scalar
zwitterionic structure with a H‚‚‚N distance of 1.11 Å requires the
increased acidity of the aspartic acid dimer.
1H-15N coupling constant of -76 Hz. Thus, a
‡Freie Universita ¨t Berlin.
†University of CaliforniasDavis.
§St. Petersburg State University.
Scheme 1.15N-Labeled Cofactor15N-PLP (internal aldimine in the
active site of AspAT, structure adapted from ref 5b)
Published on Web 07/12/2007
9558 9 J. AM. CHEM. SOC. 2007, 129, 9558-9559
10.1021/ja0728223 CCC: $37.00 © 2007 American Chemical Society
It follows that the pyridine ring and the ASP222 in the active
site of the enzyme behave as in polar organic media: when they
lose their water shell and come in direct contact, their combined
basicity leads to a high pKafor the binuclear base. By contrast, the
pKavalues of the pyridine N and the aspartate carboxylic acid in
water are not appropriate for determination of the position of the
proton in the intermolecular ASP222/pyridine N OHN hydrogen
bond. The enzyme must provide additional interactions to allow
proton transfer to the pyridine N. The hydrogen bonds from ASP222
to HIS143 and two conserved water molecules in the AspAT active
site are, therefore, probably the most important secondary interac-
tions required to shorten the H‚‚‚N distance and produce active
enzyme. This work also demonstrates that the active site environ-
ment is better modeled using proton donors in polar aprotic solvents
than in water. This is a satisfying result since highly structured
hydrogen bonding within a polar aprotic milieu lacking bulk water
is observed in enzyme active site structures.
In conclusion, we have shown that liquid and solid state15N
NMR of enzymes in conjunction with model studies provides a
powerful tool for localizing mechanistically critical protons in
enzyme active sites.
Acknowledgment. We thank the Deutsche Forschungsgemein-
schaft, Bonn, the Fonds der Chemischen Industrie, Frankfurt, and
the National Institutes of Health (Grant GM54779 to M.D.T.) for
financial support. We also thank Prof. Klaus Weisz, Greifswald,
for help with the measurement of the spectrum in Figure 1c.
Supporting Information Available: Material and methods are
presented. Shown are the1H NMR spectra corresponding to Figure 1f.
This material is available free of charge via the Internet at http://
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AspAT, (b) microcrystalline maleate-liganded holo-AspAT, and (c) holo-
AspAT in aqueous solution, (d) aldimine model in aqueous solution from
ref 7c. (e and f) Aldimine model in polar aprotic liquid in the absence and
presence of protected aspartic acid (Boc-Asp-OtBu). Distances estimated
by NMR are listed in Table 1. For the values of ?r, see ref 7e.
15N NMR spectra of15N-PLP samples: (a) microcrystalline holo-
Intermolecular OHN Hydrogen Bonds of the Systems in Figure 1
15N NMR Parameters and Geometries of the
174 ( 8.0 1.09 ( 0.05 1.54 ( 0.05 2.63 ( 0.1
180 ( 8.0 1.11 ( 0.05 1.49 ( 0.05 2.60 ( 0.1 microcryst.
167.4 ( 0.6 1.07 ( 0.01 1.64 ( 0.01 2.71 ( 0.02
262.5( 0.1 1.74 ( 0.02 1.01 ( 0.02 2.75 ( 0.04
277.4 ( 0.1
237.7 ( 0.1 1.43 ( 0.01 1.11 ( 0.01 2.54 ( 0.02
180.8 ( 0.1 1.11 ( 0.01 1.48 ( 0.01 2.59 ( 0.02
aRef external solid15NH4Cl.bAverage distances estimated from δ(15N)
according to refs 7a and 7b; possible systematic errors are not included.
cCalculated assuming linear H-bonds.dX-ray:5brON ) 2.64(74) Å.eX-
ray:5brON) 2.58(68) Å.fFreon mixture CDF3/CDF2Cl, dielectric constant
gTolylaldenamine complexes with Boc-Asp-OtBu.
C O M M U N I C A T I O N S
J. AM. CHEM. SOC. 9 VOL. 129, NO. 31, 2007 9559