A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole

Subscriber access provided by RYERSON UNIV

Article 17

Solid-State O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases Aaron W. Tang, Xianqi Kong, Victor Terskikh, and Gang Wu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08798 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Solid-State 17O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases

Aaron W. Tang,1 Xianqi Kong,1 Victor Terskikh,1,2 and Gang Wu1*

1

Department of Chemistry, Queen’s University, 90 Bader Lane,

Kingston, Ontario, Canada K7L 3N6; 2Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5

Running title: Solid-State 17O NMR of Unstable Acyl-Enzymes

*Corresponding author: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 40

Abstract We report preparation, trapping, and solid-state

17

O NMR characterization of three

unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]benzoylchymotrypsin, trans-o-methoxy-[17O]cinnamoyl-chymotrypsin, and trans-p-methoxy[17O]cinnamoyl-chymotrypsin. We show that both the

17

O chemical shifts and nuclear

quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental 17O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C=17O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the

17

O chemical shift and quadrupole coupling constant

display distinctly different sensitivities toward different aspects of hydrogen bonding such as hydrogen bond distance and direction. This work demonstrates the utility of 17O as a useful nuclear probe in NMR studies of enzymes.

Keywords: 17O NMR, acyl-enzyme, chymotrypsin, oxyanion hole, hydrogen bonding


Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Oxygen is one of the most common elements in organic and biological molecules, but remains largely inaccessible as a nuclear probe in NMR studies of these molecular systems. While the exceedingly low natural abundance (0.037%) of the only NMR-active oxygen isotope, 17O, poses some challenges, the primary obstacle of 17O NMR studies is the intrinsic quadrupolar nature of the

17

O nucleus (spin-5/2). In general, NMR spectra

for quadrupolar nuclei have significantly lower resolution than those from more conventional spin-1/2 probes such as 1H,

13

C and

15

N. However, recent advances have

shown that, with the availability of ultrahigh magnetic fields (e.g., 21 T),

17

O NMR is

beginning to become applicable in studies of biological macromolecules in both solution and solid state.1-9 In this work, we explore the utility of solid-state

17

O NMR at 21.1 T as a new

technique for studying unstable intermediates formed in enzymatic reactions. In particular, we set out to investigate whether 17O NMR can be used to probe the hydrogen bonding interactions between the substrate and protein backbone in the so-called oxyanion hole of a model enzyme, chymotrypsin (26 kDa); see Scheme 1. Chymotrypsin belongs to a family of enzymes known as serine proteases, named for a catalytically active nucleophilic serine residue in the active site. For chymotrypsin, the three catalytic residues known as the “catalytic triad”, Ser195, His57, and Asp102, form a hydrogenbond network at the active site.10,11 This hydrogen-bond network activates Ser195 for nucleophilic attack on the substrate. It is well-established that the serine protease catalyzes the hydrolysis reaction in two stages: first, the acylation step where the substrate is covalently bonded to Ser195 to form the acyl-enzyme intermediate, breaking



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the amide or ester bond and releasing part of the substrate with the free amino terminus, and second, the deacylation step where the ester bond in the acyl-enzyme intermediate is hydrolyzed. When the substrate contains a suitably stable leaving group, the formation of the acyl-enzyme intermediate occurs rapidly and the hydrolysis of the acyl-enzyme is rate-limiting.12 In addition to the “catalytic triad”, another common feature in serine proteases is the presence of the oxyanion hole.13 As seen in Scheme 1, the oxyanion hole, first identified in the pioneering work of Henderson,14 consists of two hydrogen bond donors from the backbone of Ser195 and Gly193, that stabilize the oxygen on the scissile amide/ester bond in the tetrahedral transition state. The oxyanion hole is important for both the acylation and deacylation steps. The contribution of the oxyanion hole to catalysis is significant, as enzymes with the catalytic triad disabled via site-directed mutagenesis still retain hydrolytic activity with a reaction rate a thousand-fold higher than that for the uncatalyzed hydrolysis.15,16 Inspired by previous resonance Raman spectroscopic studies of serine proteases that showed a correlation between the C=O stretching frequency and acyl-enzyme deacylation rate,17-20 we hypothesize that

17

O NMR should be a sensitive probe to the hydrogen

bonding interaction at the carbonyl oxygen atom of the substrate which is located at the center of the oxyanion hole. Our general strategy consists of two components. First, we chose to use substrates that are known to form acyl-enzyme intermediates with relatively slow deacylation rates. Second, once the acyl-enzyme intermediates are formed in aqueous solution, we attempt to trap them by quickly freeze drying the solution and then perform solid-state 17O NMR measurements.


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2. Experimental section 2.1 Synthesis of 17O-labeled N-acyl imidazole substrates Three N-acyl-imidazoles (Scheme 2) were prepared by coupling imidazole with the 17

O-labeled carboxylic acids using N,N′-carbonyldiimidazole (CDI). Synthetic details for

preparation of

17

O-labeled carboxylic acid precursors are provided in the Supporting

Information. p-N,N-Dimethylamino-[17O]benzoylimidazole (DAB-Im). This compound was synthesized following a method described previously.21 p-N,N-dimethylamino[17O2]benzoic acid (100.0 mg, 0.6054 mmol) and CDI (148.6 mg, 0.9164 mmol, 1.514 mol equiv) were added to a 10 mL round bottom flask, which was then capped with a rubber septum. The headspace was purged with nitrogen gas with a needle for 1 minute, and anhydrous THF (3 mL) was added with a syringe and needle. Some insoluble grey/blue solids remained. The headspace was again purged with nitrogen gas for 5 minutes. The reaction mixture was stirred for 19 hours, after which insoluble solid remained visible. The solvent was removed under reduced pressure until a light-grey oil and solid remained. The residue was transferred to an extraction funnel with CHCl3 (2 × 2 mL). The organic layer was washed with NaHCO3 solution (2 × 2 ml 0.8% w/v), saturated Na2SO4 solution (3 mL), then dried (Na2SO4). The organic solvent was removed under reduced pressure until a light grey/purple solid remained. The residue was dissolved in CHCl3 (2 mL), and some activated charcoal was added. The mixture was filtered through a Pasteur pipette with a cotton plug, packed with a layer of celite. The pipette was rinsed with CHCl3 (2 mL, 1 mL). The eluent was light purple, and the solvent was removed under reduced pressure. The off-white residue was dried under vacuum 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 40

overnight. The residue was dissolved in acetone (1.5 mL) and precipitated with cold H2O (8 mL), forming a white, flocculent solid. The solid was collected via suction filtration, and washed with cold H2O (2 × 4 mL) and hexanes (2 × 2 mL) to yield 47.5 mg (36.5%) of final product. 1H NMR (400 MHz, acetone-d6) δ 8.09 (s, 1H), 7.77 (dt, J = 9.2, 2.2 Hz, 2H), 7.58 (t, J = 1.38 Hz, 1H), 7.10 (d, J = 0.5 Hz, 1H), 6.87 (dt, J = 9.1, 2.2 Hz, 2H), 3.14 (s, 6H); 17O NMR (54.1 MHz, acetone-d6) δ 410.4 (br). trans-o-Methoxy-[17O]cinnamoylimidazole

(oMC-Im).

trans-o-Methoxy-

[17O2]cinnamic acid (200 mg, 1.12 mmol) and CDI (303 mg, 1.87 mmol, 1.67 mol equiv) were added to a 10 mL round bottom flask. Anhydrous THF (4 mL) was added and the flask was sealed with a rubber septum stopper. The headspace of the flask was purged with nitrogen gas introduced with a needle. The reaction mixture was stirred for 13 hours. The solvent was evaporated under reduced pressure. Crude product was dissolved in DCM (5 mL) and toluene (15 mL), and washed with NaHCO3 solution (2.5 mL 0.8% w/v), saturated Na2SO4 solution (3 × 3 mL). The organic layer was dried (Na2SO4), then the solvent was removed to yield a crude solid product. The residue was recrystallized from acetone (14 mL) and cold H2O (50 mL), then washed with cold H2O (3 × 10 mL) to yield 181 mg (70.7%) of final product. 1H NMR (500 MHz, acetone-d6) δ 8.54 (s, 1H), 8.38 (d, J = 15.6 Hz, 1H), 7.92 (dd, J = 7.7, 1.6 Hz, 1H), 7.82 (t, J = 1.3 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.50 (ddd, J = 8.7, 6.9, 1.6 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.10 (s, 1H), 7.05 (t, J = 7.57 Hz, 1H), 3.99 (s, 3H);

17

O NMR (67.7 MHz, acetone-d6) δ 384.5

(br). trans-p-Methoxy-[17O]cinnamoylimidazole (pMC-Im). The synthesis of this compound was also described previously.21 trans-p-Methoxy-[17O2]cinnamic acid (200


Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mg, 1.12 mmol) and CDI (303 mg, 1.87 mmol, 1.67 mol equiv) were added to a 10 mL round bottom flask. Anhydrous THF (4 mL) was added and the flask was sealed with a rubber septum stopper. The headspace of the flask was purged with nitrogen gas introduced with a needle. The reaction mixture was stirred for 13 hours. The solvent was evaporated under reduced pressure. Crude product was dissolved in DCM (5 mL) and toluene (15 mL), and washed with NaHCO3 solution (2.5 mL 0.8% w/v), saturated Na2SO4 solution (3 × 3 mL). The organic layer was dried (Na2SO4), then the solvent was removed to yield a crude solid product. The residue was recrystallized from acetone (14 mL) and cold H2O (50 mL), then washed with cold H2O (3 × 10 mL) to yield the final product. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 8.06 (d, 1H, J = 15.5 Hz), 7.63-7.65 (d+s, 3H), 7.10 (s, 1H), 6.98 (d, 2H, J = 6.6 Hz), 6.95 (d, 1H, J = 15.3 Hz), 3.90 (s, 3H); 17

O NMR (67.7 MHz, acetone-d6) δ 352.9 (br).

2.2 Chymotrypsin Activity Assay The activity of α-chymotrypsin (from bovine pancreas, 3 × crystallization, essentially salt free, purchased from Sigma Aldrich) was measured with a chymotrypsin activity assay adopted from the testing protocol of Sigma-Aldrich,22 where the enzyme-catalyzed hydrolysis of N-benzoyl-L-tyrosine ethyl ester (BTEE) was tracked via UV-vis spectroscopy. The 1.18 mM BTEE solution was prepared by dissolving 18.5 mg BTEE in 31.7 mL methanol, and filled to the mark with water in a 50 mL volumetric flask. A 2 M CaCl2 solution was prepared by dissolving 221.96 mg/mL anhydrous CaCl2. 1 mM HCl solution was prepared by serial dilution from concentrated HCl. The enzyme solution was prepared by dissolving enough α-chymotrypsin (approximately 30-80 µg) in 1 mM HCl to reach approximately 2-5 units/mL (1 unit is defined as 1 µmole BTEE consumed per



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 40

minute at pH 7.80 at 25°C). Then 1.420 mL of Tris buffer (80 mM, pH 7.80), 1.400 mL of BTEE, and 80 µL of CaCl2 solutions were mixed in a quartz cuvette and equilibrated at 25°C. 100 µL of the enzyme solution was added to the cuvette, which was inverted five times. UV-vis spectra were recorded immediately with a JASCO J-815 CD spectrometer using time course measurement at 256 nm over 3-5 minutes (slit width = 2500 nm).

2.3 Preparation, ESI-MS characterization, and kinetic measurement of acyl-enzyme intermediates p-N,N-Dimethylamino-[17O]benzoyl-chymotrypsin

(DAB-CHT).

α-

Chymotrypsin (50.1 mg) was dissolved in acetate buffer (1 mL 0.1 M, pH 4.00) in a 2.5 mL microcentrifuge tube. p-N,N-dimethylamino-[17O]benzoylimidazole (4.4 mg, 10 mol equiv) was dissolved in acetonitrile (100 µL). The two solutions were mixed and allowed to react for 30 minutes. Four 1-mL plastic syringes were plugged with glass wool and packed with Sephadex G-25. The reaction mixture was split into four 275 µL fractions and added to the syringes, which were then centrifuged briefly. The gel filtration process successively removed the excess free substrates, which was confirmed by solution

17

O

NMR. The eluent fractions were combined and immediately frozen in a dry ice/acetone bath, and lyophilized overnight to yield 39.1 mg (77%) of solid product. trans-o-Methoxy-[17O]cinnamoyl-chymotrypsin (oMC-CHT). α-Chymotrypsin (25.3 mg) was dissolved in acetate buffer (1 mL 0.1 M, pH 4.00) in a 2.5 mL microcentrifuge tube. trans-o-methoxy-[17O]cinnamoylimidazole (3.8 mg) was dissolved in acetonitrile (165.2 µL). 100 µL (10 mol equiv) of the above solution was added to the enzyme and allowed to react for 1 minute. Following the same procedure of running the


Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Sephadex G-25 column and lyophilization as described earlier, we obtained 20.6 mg (81%) of solid product. trans-p-Methoxy-[17O]cinnamoyl-chymotrypsin (pMC-CHT). α-Chymotrypsin (25.1 mg) was dissolved in acetate buffer (1 mL 0.1 M, pH 4.00) in a 2.5 mL microcentrifuge tube. trans-p-methoxy-[17O]cinnamoylimidazole (2.8 mg, 12 mol equiv) was dissolved in acetonitrile (100 µL). The two solutions were mixed and allowed to react for 1 minute. Following the same procedure of running the Sephadex G-25 column and lyophilization as described earlier, we obtained 20.1 mg (80%) of solid product. Mass spectrometry. Formation of covalently bonded acyl-enzymes was confirmed via electrospray ionization ion-trap mass spectrometry (ESI-MS). Mass spectra were recorded on a Thermo Scientific Orbitrap Velos Pro mass spectrometer with ESI in the positive ion mode. Spectral deconvolution was performed using Thermo Scientific ProMass, under positive ion mode with an adduct ion mass of 1.0079 Da. The output mass range was set to 20,000 Da to 80,000 Da, the default setting for large proteins. Kinetic measurement of deacylation. Deacylation rates of the acyl-enzymes were followed using UV-Vis spectroscopy23,24 on a JASCO J-815 CD spectrometer. The acylenzyme solution was prepared by dissolving 1.5 mg lyophilized acyl-chymotrypsin intermediate in 3 mL buffer (80 mM Trizma, 50 mM CaCl2, pH 7.80) in a quartz cuvette. UV-vis spectrum was recorded over wavelengths of 250-350 nm (slit width = 100 µm, data pitch = 0.1 nm, scanning speed = 100 nm/min), with sample cell kept at 25.0 °C. The data collection time points ranged between a period of 120 hours for DAB-CHT, 210 minutes for oMC-CHT, and 10 minutes for pMC-CHT. The last reading for each inhibitor is treated as the baseline against which a difference spectrum can be calculated,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

as enough time has passed to assume that hydrolysis of the acyl-enzyme complex is complete. Spectroscopic data for deacylation are provided as the Supporting Information.

2.4 Solid-state 17O NMR Solid-state 17O NMR experiments were performed on a Bruker Avance-II 900 (21.1 T) NMR spectrometer. A rotor-synchronized Hahn-echo sequence was used for MAS experiments to eliminate the acoustic ringing from the probe. A 3.2-mm Bruker HX MAS probe was used on which the effective 90º pulse for the

17

O central transition (CT) was

1.0 µs. A liquid H2O sample was used for both RF power calibration and

17

O chemical

shift referencing (δ = 0 ppm). All spectral simulations were performed with DMfit.25

2.5 Computational details Quantum chemical calculations for NMR parameters were performed with Gaussian 0926 on High Performance Computing Virtual Laboratory (HPCVL) servers. The calculations were performed using the Becke-3-Parameter, Lee-Yang-Parr (B3LYP) exchange functional and a 6-311++g(d,p) basis set. The experimental solid-state

17

O

NMR results obtained for p-methoxy-[17O]cinnamate methyl ester (pMC-Me) were used to calibrate the

17

O nuclear quadrupolar moment (Q = –2.305 fm2) as demonstrated

previously.27,28 The computed

17

O magnetic shielding constants (σ) were converted to

chemical shifts (δ) using δ = 270.2 ppm – σ. The experimental and computed 17O NMR parameters for pMC-Me are provided in Supporting Information.

3. Results and discussion


Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As acyl-enzyme intermediates are generally unstable in aqueous solution, our strategy was to quickly freeze-dry the reaction solution so that the acyl-enzymes can be trapped in the solid state. The deacylation of acyl-enzymes is known to occur very slowly in the solid state.14 To confirm the identity of the solid acyl-enzyme products formed between α-chymotrypsin and the three N-acyl imidazole substrates shown in Scheme 2, we analyzed them via electrospray ionization-ion trap mass spectrometry (ESI-MS). As seen from Figure 1, the major peaks in the ESI mass spectra of DBA-CHT, oMC-CHT and pMC-CHT appear at 25597.2 ± 1.5, 25608.9 ± 0.3, and 25609.7 ± 0.6 Da, respectively. The parent enzyme peak was found to be at 25449.2 ± 0.7 Da, which corresponds to the α1-isoform of CHT.29,30 The mass differences between the acylenzyme and the free enzyme match those expected for the three DAB (147.2 Da), oMC (160.2 Da) and pMC (160.2 Da) moieties. It is also interesting to note that, in each case, both the acyl-CHT and free CHT major peaks are flanked by satellite peaks. The MS satellite peaks having higher masses than the free CHT and acyl-CHT are clearly due to different Na+ adduct ions because of the mass separation of a multiple of 22 Da. The MS peaks below the base peaks of CHT and acyl-CHT appear at M-18 and can be attributed to the presence of a minor isoform of the enzyme, δ-CHT, which has a theoretical molecular mass of 25,430.9 Da.30 The α1- and δ-isoforms of CHT differ only in that the peptide bond of Thr147 is hydrolyzed in the α1-isoform during activation from chymotrypsinogen A, resulting in a mass difference of a water molecule (18 Da). The presence of multiple isoforms in commercial preparations of α-CHT has been previously demonstrated.29,30



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

We should note that the co-existence of α1- and δ-isoforms of CHT in our samples does not affect the conclusion of the present study because the minute difference between the two isoforms does not markedly alter the overall properties of the protein, nor does it eliminate the catalytic activity of the enzyme.31,32 More importantly, the goal of the present work is to study the hydrogen bonding interaction in the oxyanion hole of chymotrypsin, and these two isoforms are identical in this aspect. As also seen from Figure 1, the very weak peaks (> 25,700 Da) are likely due to some protein impurities, but they are not identified at this time. On the basis of the peak areas of the acyl-CHT and free CHT signals, we estimated the level of acylation to be in a range from 79% to 92% for the three acyl-CHT products; see Table 1. These are very satisfactory results. Now that the integrity of the solid acyl-enzyme samples is established, the solid-state 17

O NMR spectra can then be attributed with certainty to the trapped acyl-enzyme

intermediates. Each of the 17O MAS NMR spectra shown in Figure 2 consists of a central signal flanked by two weak spinning sidebands. These 17O NMR signals can be properly modeled by considering both the second-order quadrupolar and magnetic shielding anisotropy interactions. The

17

O NMR parameters obtained from such spectral analyses

are summarized in Table 1, together with the kinetic data measured for the three acylenzymes. It is interesting to note that the deacylation rate constant (k3) is increased from DAB-CHT to pMC-CHT by 1000-fold. Our k3 values are comparable to the literature data reported for the same acyl-enzymes.33,34 We should point out that, because the experimental values on the 17O chemical shift anisotropy (i.e., the CS tensor components) are far less reliable than the isotropic chemical shift values, in the discussion that follows


Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


we will discuss only the isotropic chemical shift (δiso) and quadrupole coupling constant (CQ). As seen from Table 1, the values of δiso and CQ observed for acyl-enzymes are generally consistent with those previously reported for the ester functional group.35,36 Among the three acyl-enzymes, we found that the following trend in the isotropic

17

O

chemical shifts (δiso): DAB-CHT > oMC-CHT > pMC-CHT. The same trend also holds for CQ. More interestingly, both of the 17O NMR parameters appear to be correlated with the deacylation rate constants measured in aqueous solution for these acyl-enzymes, as shown in Table 1. That is, the smaller the k3, the larger the value of δiso (or CQ). As far as the

17

O NMR parameters are concerned, numerous previous studies of carbonyl

compounds have firmly established that, for the oxygen atom involved in a hydrogen bond, its δiso and CQ values decrease with the increase in the hydrogen bonding strength.37-48 Thus the observed trends in δiso and CQ among the three acyl-enzyme intermediates suggest that the hydrogen bonding strength is in the following order: DABCHT < oMC-CHT < pMC-CHT. Previous Raman spectroscopic studies of acylchymotrypsin complexes17-20 suggested that a stronger hydrogen-bonding environment in the oxyanion hole provides greater transition state stabilization, leading to a higher deacylation rate of the acyl-enzyme. Therefore, our solid-state

17

O NMR results for

DAB-CHT, oMC-CHT, and pMC-CHT are consistent with this view. To further investigate the relationship between

17

O NMR parameters and hydrogen

bonding interactions in the oxyanion hole, we performed extensive quantum chemical computations. First, to rule out the possibility that the observed

17

O NMR parameters

reflect only the electronic effects from different acyl moieties, we computed


17

O NMR


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 40

parameters for three ethyl ester analogs where the acyl moieties of DAB, oMC, and pMC are maintained. The computational results listed in Table 2 show that the three analogs exhibit very similar 17O NMR parameters. For example, the values of δiso and CQ for the three ethyl ester analogs differ by only 4 ppm and 0.2 MHz, respectively. These results strongly suggest that it is necessary to model the hydrogen bonding interaction in the oxyanion hole for the acyl-enzymes. Before we build a computational model to mimic the hydrogen bonding environment in the oxyanion hole of chymotrypsin, we surveyed the crystallographic data for acyl-enzymes of closely related serine proteases currently available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). After a careful examination of the structural features, we identified the following two structural parameters as the major variables that have the biggest impact on the placement of the carbonyl oxygen atom in the oxyanion hole: the Cβ-Oγ-C1 bond angle (θ) and the Cα-Cβ-Oγ-C1 dihedral angle (φ), as shown in Figure 3. The surveyed data set is listed in Table 3. Among the systems examined, the bond angle θ ranges from 114° to 131° and the dihedral angle φ ranges from 83° to 110°. As a result, the two O···N hydrogen bond distances in the oxyanion hole can vary between 2.45 and 3.46 Å. This is essentially the whole range of the O···N hydrogen bond. Table 3 also listed the values of two additional torsional angles, Θ193 and Θ195, that define the directions of the two hydrogen bonds in the oxyanion hole (vide infra). We chose to use the crystal structure of trans-2,4-dihydroxycinnamoyl-γchymotrypsin (PDB ID: 1K2I)51 to construct our acyl-enzyme model because the cinnamoyl moiety in 1K2I is structurally similar to the acyl groups used in this study. In addition, γ-chymotrypsin, being a complex of α-chymotrypsin and its autolysis products,


Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shares the identical primary sequence with α-chymotrypsin.58 To reduce the computational load, only amino acid residues from 192 to 196 were used in the quantum chemical calculation to emulate the environment surrounding the oxyanion hole, as shown in Figure 3. Those residues were chosen to extend one residue beyond Gly193 and Ser195, whose backbone hydrogen atoms are responsible for the hydrogen bonding in the oxyanion hole. The C-terminus of Gly196 is terminated with N-methylamide, with the amide nitrogen and the methyl carbon occupying the same position as the backbone N and Cα of Gly197 to mimic neighboring peptide backbones. Likewise, the N-terminus of Met192 is terminated with an acetyl group, with the carbon atoms occupying the same positions as the backbone carbonyl carbon and Cα of Cys191. The trans-2,4dihydroxycinnamoyl acyl group in 1K2I was replaced with an O-acetyl group to reduce the computational cost. Using this model, we examined how 17O NMR parameters change as a function of the two aforementioned structural variables: θ and φ. The bond angle θ was varied systematically from 100° to 130°, and the dihedral angle φ was varied from 80° to 120°, both in 5° increments, to produce a total of 63 θ-φ combinations. Before calculating the

17

O NMR parameters for each θ-φ pair, a partial geometry optimization

was performed with the three atoms involved in the hydrogen bonding in the oxyanion hole (the two amide hydrogen atoms from the protein backbone Ser195 and Gly193, and the carbonyl oxygen, O1) allowed to move freely, while the remaining atoms were frozen in place. This permits the O atom and the hydrogen bonded H atoms to explore local energy minima without interference from the rest of the model. The calculated and δiso are displayed in Figure 4 as contours in the θ-φ space.


17

O CQ


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 40

It can be seen immediately from Figures 4(a) and 4(b) that, within the ranges of θ and φ examined, the 17O NMR parameters of the acyl-enzyme model display significant changes: CQ by 1 MHz and δiso by 55 ppm. These changes clearly reflect the changing degree of hydrogen bonding within the oxyanion hole. In Figures 4(a) and 4(b), we also indicate where the real crystal structures of the acyl-enzymes listed in Table 3 would appear on the 2D contour maps. One striking observation is that CQ and δiso display different responses to the hydrogen bonding change. That is, while CQ shows a minimum at (θ = 112°, φ = 80°), the minimum δiso value occurs at around (θ = 123°, φ = 95°). To better decipher the relationship between the

17

O NMR parameters (CQ and δiso) and

hydrogen bonding, it is also important to evaluate how the two H-bond distances in the model, O···N(Ser195) and O···N(Ser193), would change in the same θ-φ space. These are shown in Figures 4(c) and 4(d) where two general trends can be seen immediately. (1) Decreasing the Cα-Cβ-Oγ-C1 dihedral angle, φ, turns the carbonyl oxygen atom towards the oxyanion hole, moving closer to both hydrogen bond donors. (2) Decreasing θ from 130° to 100° rotates the carbonyl oxygen towards N(Ser195), thus shortening the O···N(Ser195) distance, but has little effect on the O···N(Gly193) distance. Now inspection of Figure 4 reveals a remarkable similarity between Figures 4(a) and 4(c). This means that CQ appears to be determined primarily by the O···N(Gly193) distance. At the same time, however, it remains unclear from Figures 4(c) and 4(d) why the landscape of δiso should be different. To further investigate the puzzling results on δiso in Figure 4(b), we now consider another important aspect of hydrogen bonding: the hydrogen bond direction. In a recent survey of enzymes containing an oxyanion hole and small molecule analogs, Simón and


Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Goodman59 noted that enzymes often contain hydrogen bonds that deviate from an arrangement that allows for ideal (or maximum) hydrogen bonding interactions. Specifically, they found that, for enzymes, the N···O1=C1-Oγ dihedral angles, Θ (as defined in Figure 3), are more frequently found to be in a range closer to 90° (where the hydrogen bond is “out-of-the plane”), than being “in-plane” (Θ ≈ 0°), which is commonly seen in the crystal structures of small molecules. This sub-optimal hydrogen bonding arrangement in enzymes is theorized to allow for stabilization of the transition state without stabilizing the ground state reactant, which would hinder the catalysis provided by the enzyme.57 Now using Θ as a measure of the hydrogen bond direction, we examined the acyl-enzymes with known crystal structures (see Table 3). First we noticed that Θ195 values are narrowly distributed around 40° (meaning the N···O hydrogen bond being in the “out-of-the-plane” mode), but Θ193 values span a much larger range. The former feature is clearly due to the fact that the access of the HN(Ser195) to the acyl C=O, which is attached to the Oγ of the same residue, Ser195, is highly restricted. We further discovered that the acyl-enzymes close to the δiso minimum (e.g., B, Da, H) have the O···N(Gly193) hydrogen bond largely in the “out-of-the-plane” mode whereas those close to the CQ minimum (e.g., Db, F) are in the “in plane” mode. This analysis then suggests that, within the narrowly defined acyl-enzymes studied here, CQ is primarily determined by the O···N(Gly193) hydrogen bond distance and δiso is more sensitive to the O···N(Gly193) hydrogen bond direction. Another interesting observation from Figure 4 is that the acyl-enzymes with known crystal structures (labeled A through J) are clustered around the δiso minimum. This leads us to hypothesize that, between CQ and δiso, the latter is a more direct measure of the hydrogen bonding environment in the oxyanion



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

hole of serine proteases. As we have established a correlation between δiso and deacylation kinetics, it is tempting to generalize this idea to link δiso to the catalytic efficiency of serine proteases. Of course, the two aspects of hydrogen bonding, distance and direction, are often coupled to each other. The complex relationships between the 17O NMR parameters (CQ and δiso) and hydrogen bonding geometry within the oxyanion hole make quantitative interpretation of the experimental 17O NMR data difficult. Nonetheless, the results presented here serve as a promising start for further experimental and computational studies.

4. Conclusions We have carried out a solid-state

17

intermediates. We found that both the

O NMR study for three unstable acyl-enzyme 17

O isotropic chemical shift and quadrupole

coupling constant obtained from solid-state NMR are correlated to the deacylation rate constant of the acyl-enzymes measured in aqueous solution. This may suggest that the deacylation kinetics of acyl-enzymes is determined mainly by the hydrogen bonding interaction that the carbonyl oxygen atom experiences in the oxyanion hole. This interpretation is consistent with the conclusions drawn from previous resonance Raman spectroscopic studies. Our quantum mechanical calculations for an acyl-enzyme model generated further insight into the relationship between

17

O NMR parameters and the

hydrogen bonding interaction in the oxyanion hole of series proteases. In particular, we demonstrated that both hydrogen bond distance and direction have significant impacts on the

17

O chemical shift and quadrupolar coupling constant. The computations confirmed

that the variations in

17

O NMR parameter observed experimentally for the three acyl-


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enzyme intermediates are due to the different hydrogen bonding environments in the oxyanion hole. The present study demonstrates the utility of solid-state

17

O NMR in

studying a model serine protease. We believe that not only can this work be extended to other serine proteases, but solid-state 17O NMR in general can provide unique insight into enzyme kinetics and mechanisms.

Acknowledgement This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada. Access to the 900 MHz NMR spectrometer was provided by the National Ultrahigh Field NMR Facility for Solids (Ottawa, Canada), a national research facility funded by a consortium of Canadian universities, National Research Council Canada

and

Bruker

BioSpin

and

managed

by

the

University

of

Ottawa

(http://nmr900.ca). We thank Dr. Jiaxi Wang for assistance in recording ESI-MS spectra for acyl-enzymes.

Supporting Information Synthesis of 17O-labeled carboxylic acids. Kinetic data for deacylation of acyl-enzymes. Experimental and simulated solid-state

17

O NMR spectra of p-methoxycinnamic acid

methyl ester. Full citation of ref.26.

References 1. Wu, G. Solid-state 17O NMR studies of organic and biological molecules. Prog. Nucl. Magn. Reson. Spectrosc. 2008, 52, 118-169. 2. Wong, A.; Poli, F. Solid-state 17O NMR studies of biomolecules. Annu. Rep. NMR Spectrosc. 2014, 83, 145-220.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40

3. Gerothanassis, I. P. Oxygen-17 NMR spectroscopy: Basic principles and applications. Part I. Prog. Nucl. Magn. Reson. Spectrosc. 2010, 56, 95-197. 4. Gerothanassis, I. P. Oxygen-17 NMR spectroscopy: Basic principles and applications. Part II. Prog. Nucl. Magn. Reson. Spectrosc. 2010, 57, 1-110. 5. Wu, G. Solid-state 17O NMR studies of organic and biological molecules: Recent advances and future directions. Solid State Nucl. Magn. Reson. 2016, 73, 1-14. 6. Zhu, J.; Kwan, I. C. M.; Wu, G. Quadrupole-central-transition 17O NMR spectroscopy of protein-ligand complexes in solution. J. Am. Chem. Soc. 2009, 131, 14206-14207. 7. Zhu, J.; Ye, E.; Terskikh, V.; Wu, G. Solid-state 17O NMR spectroscopy of protein-ligand complexes. Angew. Chem. Int. Ed. 2010, 49, 8399-8402. 8. Zhu, J.; Wu, G. Quadrupole central transition 17O NMR spectroscopy of biological macromolecules in aqueous solution. J. Am. Chem. Soc. 2011, 133, 920-932. 9. Young, R. P.; Caulkins, B. G.; Borchardt, D.; Bulloch, D. N.; Larive, C. K.; Dunn, M. F.; Mueller, L. J. Solution-state 17O quadrupole central-transition NMR spectroscopy in the active site of tryptophan synthase. Angew. Chem. Int. Ed. 2016, 55, 1350-1354. 10. Blow, D. M.; Birktoft, J. J.; Hartley, B. S. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 1969, 221, 337-340. 11. Hedstrom, L. Serine protease mechanism and specificity. Chem. Rev. 2002, 102, 4501-4524. 12. Hartley, B. S.; Kilby, B. A. The reaction of p-nitrophenyl esters with chymotrypsin and insulin. Biochem. J. 1954, 56, 288-297. 13. Kraut, J. Serine proteases: structure and mechanism of catalysis. Ann. Rev. Biochem. 1977, 46, 331-358. 14. Henderson, R. Structure of crystalline α-chymotrypsin: IV. The structure of indoleacryloyl-α-chymotrypsin and its relevance to the hydrolytic mechanism of the enzyme. J. Mol. Biol. 1970, 54, 341-354. 15. Corey, D. R.; Craik, C. S. An investigation into the minimum requirements for peptide hydrolysis by mutation of the catalytic triad of trypsin. J. Am. Chem. Soc. 1992, 114, 1784-1790. 16. Carter, P.; Wells, J. A., Dissecting the catalytic triad of a serine protease. Nature 1988, 332, 564-568. 17. Tonge, P. J.; Carey, P. R. Direct observation of the titration of substrate carbonyl groups in the active site of alpha-chymotrypsin by resonance Raman spectroscopy. Biochemistry 1989, 28, 6701-6709. 18. Tonge, P. J.; Carey, P. R. Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity. Biochemistry 1990, 29, 10723-10727. 19. Tonge, P. J.; Carey, P. R. Forces, bond lengths, and reactivity: fundamental insight into the mechanism of enzyme catalysis. Biochemistry 1992, 31, 9122-9125. 20. Tonge, P. J.; Pusztai, M.; White, A. J.; Wharton, C. W.; Carey, P. R. Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques. Biochemistry 1991, 30, 4790-4795.


Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21. Kong, X.; Tang, A.; Wang, R.; Ye, E.; Terskikh, V.; Wu, G. Are the amide bonds in N-acyl imidazoles twisted? A combined solid-state 17O NMR, crystallographic, and computational study. Can. J. Chem. 2015, 93, 451-458. 22. Wirnt, R. Chymotrypsin. In Methods of Enzymatic Analysis, 2nd ed.; Bergmeyer, H.-U., Ed., Academic Press: New York, 1974, pp 1009-1012. 23. Bender, M. L.; Schonbaum, G. R.; Zerner, B. Spectrophotometric investigations of the mechanism of α-chymotrypsin-catalyzed hydrolyses. Detection of the acyl-enzyme intermediate. J. Am. Chem. Soc. 1962, 84, 2540-2550. 24. Schonbaum, G. R.; Zerner, B.; Bender, M. L. The spectrophotometric determination of the operational normality of an α-chymotrypsin solution. J. Biol. Chem. 1961, 236, 2930-2935. 25. Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calvé, S.; Alonso, B.; Durand, J.O.; Bujoli, B.; Gan, Z.; Hoatson, G. Modelling one- and two-dimensional solid-state NMR spectra. Magn. Reson. Chem. 2002, 40, 70-76. 26. Gaussian 09, Revision B.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A., et al. Gaussian, Inc.: Wallingford, CT, 2009. 27. Dong, S.; Ida, R.; Wu, G. A combined experimental and theoretical 17O NMR study of crystalline urea: An example of large hydrogen-bonding effects. J. Phys. Chem. A 2000, 104, 11194-11202. 28. Wu, G.; Hook, A.; Dong, S.; Yamada, K. A Solid-state NMR and theoretical study of the 17O electric field gradient and chemical shielding tensors of the oxonium ion in p-toluenesulfonic acid monohydrate. J. Phys. Chem. A 2000, 104, 4102-4107. 29. Ashton, D. S.; Beddell, C. R.; Cooper, D. J.; Green, B. N.; Oliver, R. W. A.; Welham, K. J. Some electrospray mass spectrometric evidence for the existence of covalent O-acyl enzyme intermediates. FEBS Lett. 1991, 292, 201-204. 30. Ashton, D. S.; Beddell, C. R.; Cooper, D. J.; Green, B. N.; Oliver, R. W. A.; Welham, K. J. On the purity of 3X-recrystallised bovine α-chymotrypsin. Biochem. Biophys. Res. Commun. 1993, 192, 75-81. 31. Bender, M. L.; Killheffer, J. V.; Cohen, S. Chymotrypsin. Crit. Rev. Biochem. Mol. Biol. 1973, 1, 149-199. 32. Krigbaum, W. R.; Godwin, R. W. Molecular conformation of chymotrypsinogen and chymotrypsin by low-angle x-ray diffraction. Biochemistry 1968, 7, 3126-31. 33. Whiting, A. K.; Peticolas, W. L. Details of the acyl-enzyme intermediate and the oxyanion hole in serine protease catalysis. Biochemistry 1994, 33, 552-561. 34. Bernhard, S. A.; Hershberger, E.; Keizer, J. The influence of pH on the rate of hydrolysis of acylchymotrypsins. Biochemistry 1966, 5, 4120-4126. 35. Hagaman, E. W.; Chen, B.; Jiao, J.; Parsons, W. Solid-state 17O NMR study of benzoic acid adsorption on metal oxide surfaces. Solid State Nucl. Magn. Reson. 2012, 41, 60-67. 36. Kong, X.; Shan, M.; Terskikh, V.; Hung, I.; Gan, Z.; Wu, G. Solid-state 17O NMR of pharmaceutical compounds: Salicylic acid and aspirin. J. Phys. Chem. B 2013, 117, 9643-9654. 37. Butler, L. G.; Brown, T. L. Nuclear quadrupole coupling constants and hydrogen bonding. Molecular orbital study of oxygen-17 and deuterium field gradients in formaldehyde-water hydrogen bonding. J. Am. Chem. Soc. 1981, 103, 6541-6549.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

38. Butler, L. G.; Cheng, C. P.; Brown, T. L. Oxygen-17 nuclear quadrupole double resonance. 6. Effects of hydrogen bonding. J. Phys. Chem. 1981, 85, 2738-2740. 39. Gready, J. E. The relationship between nuclear quadrupole coupling constants and the asymmetry parameter. The interplay of theory and experiment. J. Am. Chem. Soc. 1981, 103, 3682-3691. 40. Gready, J. E. Theoretical study of the variability of the electric field gradient tensor of oxygen nuclei in organic molecules. J. Phys. Chem. 1984, 88, 3497-3503. 41. Kuroki, S.; Takahashi, A.; Ando, I.; Shoji, A.; Ozaki, T. Hydrogen-bonding structural study of solid peptides and polypeptides containing a glycine residue by 17O NMR spectroscopy. J. Mol. Struct. 1994, 323, 197-208. 42. Kuroki, S.; Ando, S.; Ando, I. An MO study of nuclear quadrupolar coupling constant and nuclear shielding of the carbonyl oxygen in solid peptides with hydrogen bonds. Chem. Phys. 1995, 195, 107-116. 43. Wu, G.; Yamada, K.; Dong, S.; Grondey, H. Intermolecular hydrogen-bonding effects on the amide oxygen electric-field-gradient and chemical shielding tensors of benzamide. J. Am. Chem. Soc. 2000, 122, 4215-4216. 44. Yamada, K.; Dong, S.; Wu, G. Solid-state 17O NMR investigation of the carbonyl oxygen electric-field-gradient tensor and chemical shielding tensor in amides. J. Am. Chem. Soc. 2000, 122, 11602-11609. 45. Wu, G.; Dong, S.; Ida, R.; Reen, N. A solid-state 17O nuclear magnetic resonance study of nucleic acid bases. J. Am. Chem. Soc. 2002, 124, 1768-1777. 46. Wu, G.; Yamada, K. Determination of the 17O NMR tensors in potassium hydrogen dibenzoate: A salt containing a short O...H...O hydrogen bond. Solid State Nucl. Magn. Reson. 2003, 24, 196-208. 47. Wong, A.; Pike, K. J.; Jenkins, R.; Clarkson, G. J.; Anupold, T.; Howes, A. P.; Crout, D. H. G.; Samonson, A.; Dupree, R.; Smith, M. E. Experimental and theoretical 17 O NMR study of the hydrogen-bonding on C=O and O-H oxygens in carboxylic solids. J. Phys. Chem. A 2006, 110, 1824-1835. 48. Kwan, I. C. M.; Mo, X.; Wu, G. Probing hydrogen bonding and ion-carbonyl interactions by solid-state 17O NMR spectroscopy: G-ribbon and G-quartet. J. Am. Chem. Soc. 2007, 129, 2398-2407. 49. Dixon, M. M.; Brennan, R. G.; Matthews, B. W. Structure of γ-chymotrypsin in the range pH 2.0 to pH 10.5 suggests that γ-chymotrypsin is a covalent acyl enzyme adduct at low pH. Int. J. Biol. Macromol. 1991, 13, 89-96. 50. Yennawar, N. H.; Yennawar, H. P.; Farber, G. K. X-ray crystal structure of gamma-chymotrypsin in hexane. Biochemistry 1994, 33, 7326-7336. 51. Ghani, U.; Ng, K. K.; Atta ur, R.; Choudhary, M. I.; Ullah, N.; James, M. N. Crystal structure of γ-chymotrypsin in complex with 7-hydroxycoumarin. J Mol Biol 2001, 314, 519-525. 52. Kashima, A.; Inoue, Y.; Sugio, S.; Maeda, I.; Nose, T.; Shimohigashi, Y. X-ray crystal structure of a dipeptide-chymotrypsin complex in an inhibitory interaction. Eur. J. Biochem. 1998, 255, 12-23. 53. Wilmouth, R. C.; Edman, K.; Neutze, R.; Wright, P. A.; Clifton, I. J.; Schneider, T. R.; Schofield, C. J.; Hajdu, J. X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate. Nat. Struct. Biol. 2001, 8, 689-94.


Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


54. Katona, G.; Wilmouth, R. C.; Wright, P. A.; Berglund, G. I.; Hajdu, J.; Neutze, R.; Schofield, C. J. X-ray structure of a serine protease acyl-enzyme complex at 0.95-A resolution. J. Biol. Chem. 2002, 277, 21962-21970. 55. Radisky, E. S.; Lee, J. M.; Lu, C.-J. K.; Koshland, D. E. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc. Natl. Acad. Sci. USA 2006, 103, 6835-6840. 56. Schmidt, A.; Lamzin, V. S. Extraction of functional motion in trypsin crystal structures. Acta Crystallogr. D 2005, 61, 1132-1139. 57. Mangel, W. F.; Singer, P. T.; Cyr, D. M.; Umland, T. C.; Toledo, D. L.; Stroud, R. M.; Pflugrath, J. W.; Sweet, R. M. Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin. Biochemistry 1990, 29, 8351-8357. 58. Harel, M.; Su, C. T.; Frolow, F.; Silman, I.; Sussman, J. L. γ-Chymotrypsin is a complex of α-chymotrypsin with its own autolysis products. Biochemistry 1991, 30, 5217-5225. 59. Simón, L.; Goodman, J. M. Enzyme catalysis by hydrogen bonds: The balance between transition state binding and substrate binding in oxyanion holes. J. Org. Chem. 2010, 75, 1831-1840.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 40

Table 1. Experimental deacylation rate constants (k3),a percent acylation,b and 17O NMR tensor parameters obtained for the three acyl-enzyme intermediates. Acyl-enzyme k3 (s-1) % acylation δiso (ppm) Ω (ppm)c CQ (MHz) κd

ηQ

DAB-CHT

(6.4 ± 0.1) × 10–6

92 ± 5

323 ± 5

650 ± 80

0.2 ± 0.2

10 ± 1

0.6 ± 0.2

oMC-CHT

(2.8 ± 0.1) × 10–4

87 ± 5

319 ± 5

560 ± 80

0.2 ± 0.2

9.5 ± 0.8

0.6 ± 0.2

pMC-CHT

(8.5 ± 0.3) × 10–3

79 ± 5

288 ± 5

470 ± 80

0.2 ± 0.2

7.0 ± 0.5

0.8 ± 0.2

a

Measured in aqueous solution at pH 7.8 and 25 °C as described in the Experimental section. b Determined from ESI-MS data shown in Figure 1. c Span Ω = δ11 − δ33. d Skew κ = 3(δ22 − δiso)/Ω.

24

ACS Paragon Plus Environment

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. A summary of calculated 17O NMR parameters for acyl-enzyme analogs. The experimental 17O NMR data of p-methoxycinnamic acid methyl ester (pMC-Me) are given in parenthesis. The solid-state 17O NMR spectra of pMC-Me are provided in the supporting Information (Figure S4). Molecule DAB-Et oMC-Et pMC-Et (exptl for pMC-Me)

δiso (ppm) 329.1 330.1 326.1 (325)

Ω (ppm) 629.3 630.6 627.0 (512)

25

κ 0.64 0.68 0.67 (0.75)


CQ (MHz) 8.44 8.32 8.27 (8.20)

ηQ 0.0 0.0 0.0 (0.02)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 40

Table 3. Crystal structural data for acyl-enzyme intermediates of serine proteases reported in the literature. Structure

PDB ID

X-ray data set resolution (Å)

A

2GCT49

1.80

B

1GMC50

2.20

C

1K2I51

1.80

Da

1AB952

1.60

Db E

1HAX53

1.60

F

1GVK54

0.94

G

2AGE55

1.15

H

1XVM56

1.10

I

2AH455

1.13

J

1GBT57

2.00

rO···N, Å (Θ, N···O1=C1-Oγ, °) Ser195 Gly193 3.253 2.452 (54) (120) 3.046 2.952 (46) (140) 3.405 2.990 (47) (114) 3.459 2.986 (39) (102) 2.887 2.762 (49) (165) 2.806 2.729 (43) (174) 2.723 2.849 (28) (160) 2.855 3.016 (36) (173) 2.965 2.836 (36) (148) 3.142 3.454 (45) (104) 3.280 3.409 (43) (90)

26


θ (°)

φ (°)

131

87

119

100

120

110

127

101

117

85

124

83

117

89

120

91

121

94

118

98

114

102

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme and Figure Captions

Gly193

N Ser195

N H

H oxyanion hole

17O

C Ser195 CH O 2

R

Scheme 1. Formation and hydrogen bonding environment of the oxyanion hole in serine proteases.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H3C

17 O N H3C

N

O

17 O

17 O N

CH 3

N

N H3C

N

DAB-Im DAB-Im

oMC-Im

N

O

pMC-Im

Scheme 2. Molecular structures of 17O-labeled N-acyl imidazoles.

28


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acyl-CHT CHT

(a)

∆ = 148.0

25597.2 ± 1.5

25449.2 ± 0.7

∆ = 159.7 25608.9 ± 0.3

(b) ∆ = 160.5

25609.7 ± 0.6

(c) 25400

25450

25500

25550

25600

25650

25700

25750

25800

Mass (Da)

Figure 1. Deconvoluted ESI mass spectra of (a) DAB-CHT, (b) oMC-CHT, and (c) pMC-CHT.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

*

(b)

(c) 800

600

400

200 δ(17O)/ppm

0

-200

-400

Figure 2. Experimental (blue trace) and simulated (red trace) 17O MAS NMR spectra of (a) DAB-CHT, (b) oMC-CHT, and (c) pMC-CHT. The weak signal marked with * at 380 ppm is from the ZrO2 rotor. For each sample, approximately 20 mg of solid acyl-enzyme were packed into a 3.2 mm ZrO2 rotor. The sample spinning frequency was 20 kHz and the recycle delay of 30 ms. A total of 2.0 × 106 transients were collected for each spectrum (total experimental time ≈ 23 hr).

30


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a)

Cα Cβ

b)

C1 Oγ

Oγ Ser195

R C1 O1 H N Gly193

Θ193 = N(Gly193)

O1=C 1-Oγ

Θ195 = N(Ser193)

O1=C1-Oγ

H N Ser195

Figure 3. (a) A computational model for Oγ-acetyl-chymotrypsin. The Cα-Cβ-Oγ-C1 dihedral angle (φ) and Cβ-Oγ-C1 bond angle (θ) are defined. The two hydrogen bonds between backbone amide protons from Ser195 and Gly193 and the carbonyl oxygen atom are shown to highlight the oxyanion hole. (b) Definition of torsional angles Θ193 and Θ195 for describing the directions of the two hydrogen bonds in the oxyanion hole of acylenzymes.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a)

Page 32 of 40

b)

C J

C

B

Da

J

Da

I

I H

F Db

A

Db

c)

H

G

F

B

E

G A E

d)

Figure 4. Dependences of computed 17O NMR parameters (a: CQ in MHz; b: δiso in ppm) and hydrogen bond distances (c: O···NGly193; d: O···NSer195, Å) on the Cβ-Oγ-C1 bond angle (θ) and the Cα-Cβ-Oγ-C1 dihedral angle (φ). The filled circles (A-J) indicate the positions of the known acyl-enzymes listed in Table 3. Note that, because the acylenzyme shown in Fig. 3 does not necessarily produce the same hydrogen bond distances as the actual crystal structures, filled circles for A-J are not plotted onto (c) and (d).

32


Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graphics

Gly193

Asp194

Oxyanion hole

N H 17O

17O

NH O

C

NMR at 21 T

R

Ser195

33


Gly193 The Journal of Physical Chemistry Page 34 of 40

N

Ser195 N H H 1 2 oxyanion 3 17O 4 5 ACS Paragon C Plus Environment 6 7 R Ser195 CH2 O 8

hole

DAB-Im O

H3C H3C O O

17 O

17 O

O

PageThe 35 of Journal 40 of Physical Chemistry N

C H3C N N CH3 CH3

1 2

N N

N

N

N N

N H3C

17 O

N

ACS Paragon Plus Environment DAB-Im

oMC-Im

DAB-Im DAB-Im O

oMC-Im oMC-Im O

O

O

O

pMC-Im

pMC-Im

N

Acyl-CHT

The Journal of Physical Chemistry Page 36 of 40 CHT Δ = 148.0

25597.2 ± 1.5

1

(a) 2

25449.2 ± 0.7

3 4 Δ = 159.7 5 25608.9 ± 0.3 6 7 8 (b) 9 10 Δ = 160.5 11 25609.7 ± 0.6 12 13 (c) 14 15 ACS Paragon Plus Environment 16 25400 25450 25500 25550 25600 25650 25700 25750 17 Mass (Da) 18

25800

PageThe 37 of Journal 40 of Physical Chemistry

(a) 1 2 3 (b) 4 5 6 7 8 (c) 9 10 11 800 12 13

*

ACS Paragon Plus Environment 600

400

200 δ(17O)/ppm

0

-200

-400

a) 1 2 3 4 5 6 7 b) 8 9 10 11 12 13 14

The Journal of Physical Chemistry Page 38 of 40

Cα Cβ

Oγ

C1

Oγ Ser195

R C1

Θ193 = N(Gly193)O1=C1-Oγ Θ195 = N(Ser193)O1=C1-Oγ

O1

ACS Paragon Plus Environment H

N Gly193

H

N Ser195

Page a) 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 c) 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

The Journal of Physical b) Chemistry

C J

C

B I

Da

J

Da

I

H F Db

B H

G

F Db

A E

G A E

d)


Oxyanion hole The Journal of Physical Chemistry Page 40 of 40 Gly193

Asp194

1 2 3 4 5 6

N H

NH 17O

17O

NMR at 21 T

O Paragon C R ACS Plus Environment Ser195

A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole

Recommend Documents