Investigating Enzyme Active-Site Geometry and Stereoselectivity in an

Activity pubs.acs.org/jchemeduc

Investigating Enzyme Active-Site Geometry and Stereoselectivity in an Undergraduate Biochemistry Lab Saeed Roschdi and Theodore J. Gries* Department of Chemistry, Beloit College, Beloit, Wisconsin 53511, United States S Supporting Information *

ABSTRACT: The three-dimensional nature of interactions between enzymes and their substrates often leads to exacting spatial binding orientations and stereoselectivity in chemical catalysis. Dehydrogenases that use NAD+ as a redox cofactor tend to show stereospecificity in transferring a hydride to the C4 of the nicotinamide moiety via either the re or the si face. The stereospecificity of this hydride transfer, which results in a prochiral C4 in the reduced NADH, may be determined using deuterated substrates and 1H NMR spectroscopy. A biochemistry lab activity that combines analysis of the intermolecular interactions and spatial orientation between substrate, cofactor, and enzyme from a recent crystal structure of yeast alcohol dehydrogenase with improved in situ single-tube reaction conditions for elucidating the prochiral specificity of yeast alcohol dehydrogenase through 1H NMR spectra analysis is presented. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Inquiry-Based/Discovery Learning, Conformational Analysis, Proteins/Peptides

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prochiral specificity of YADH by NMR spectroscopy using an in situ reaction with deuterated ethanol and requiring no purification is presented. Besides eliminating multiple steps where product may be lost by students, this new activity frees course time to investigate the intermolecular interactions between and spatial orientation of YADH, substrate, and cofactor from a recent structural study and requires fewer lab resources.3 By using both deuterated and undeuterated substrates, students are able to collect results to directly compare the pro-R and pro-S chemical shifts without the instructor having to purchase and store reduced NADH. The inquiry-based activity to guide students through connecting the context of crystal structure models to wet-lab experimental results is included.

nder anaerobic growth conditions when oxygen is not present to act as a final electron acceptor, yeast alcohol dehydrogenase (YADH) catalyzes the oxidation of the reduced form of β-nicotinamide adenine dinucleotide (NADH) to maintain levels of the oxidized form of β-nicotinamide adenine dinucleotide (NAD+) within yeast cells. YADH transfers the pro-R hydride on C4 of NADH to the re face of the carbonyl carbon of acetaldehyde to generate ethanol and NAD+ in a fully reversible reaction. To study the reverse direction of this reaction under irreversible conditions in which acetaldehyde and NADH are generated, semicarbazide may be added to reactions to form an imine with the aldehyde under basic conditions (Scheme 1). Due to nicotinamide ring moiety conformation and π-stacking interactions between the nicotinamide and adenylyl moieties of NADH, the electronic environment around the pro-R and pro-S hydrogens on C4 of the nicotinamide moiety is sufficiently different and results in unique chemical shift values for each when analyzed by 1H NMR spectroscopy.1 Using completely deuterated ethanol as a substrate and protonated NAD+ as a cofactor for the irreversible reaction shown in Scheme 1, the hydrogen initially present on C4 of NAD+ can be shown to be the pro-S hydrogen of NADH, whereas the deuteride transferred to C4 of NAD+ from ethanol results in the absence of a pro-R NMR peak. Thus, YADH transfers the pro-R hydride of NADH. A previously reported undergraduate lab activity investigated the prochiral specificity of alcohol and glucose dehydrogenases using 1H NMR spectroscopy.2 The reported protocols required several purification steps before NMR spectral analyses could proceed. Herein, a single-tube method for determining the © XXXX American Chemical Society and Division of Chemical Education, Inc.

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EXPERIMENTAL PROCEDURE Ethanol-d6 (≥99.9% D), deuterium oxide (D2O; 99.9% D with 0.05 wt % TSP), semicarbazide hydrochloride (≥99%), potassium pyrophosphate (97%), ethanol (≥99.8%), sodium hydroxide (NaOH; ≥98%), β-nicotinamide adenine dinucleotide sodium salt (NAD+; ≥95%), and alcohol dehydrogenase from Saccharomyces cerevisiae (YADH; 361 U/mg) were obtained from Sigma-Aldrich (St. Louis, MO) and were used without further purification. Stock solutions of 0.96 M NAD+, 4.8 M ethanol-d 6 , 4.8 M ethanol, 1.5 M potassium pyrophosphate (pH not adjusted), 1.5 M semicarbazide, 3 M Received: October 18, 2016 Revised: May 22, 2017

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DOI: 10.1021/acs.jchemed.6b00772 J. Chem. Educ. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION Providing experiences where students connect foundational course content with laboratory techniques via drawing evidence-based conclusions from data are considered an important component of an undergraduate biochemistry curriculum. The relationship between protein structure and function has remained one critical example of a foundational course topic that requires multiple exposures throughout a curriculum to gain competency. A one-week activity that was implemented in a biochemistry research-based course and in a workshop-format upper-division undergraduate biochemistry course that meets three times each week for 2 h combined classroom and lab sessions and provides deep engagement with both enzyme active-site architecture, and 1H NMR spectral analysis is presented (Supporting Information; student and teacher versions are provided). In total, 21 students successfully completed the activity on their initial attempt. The activity connected seamlessly with content on stereoselectivity and dehydrogenases found in common biochemistry textbooks.4,5 If part of the included pre- and postlab activities is assigned outside of class time, the entire activity may be easily modified to fit into a more classical 3 h lab session. During the first day or prelab portion of this activity (Supporting Information), students • connected basic chemical knowledge of intermolecular interactions to understand the basis for substrate and cofactor interactions within the spatial orientation of YADH active-site residues • practiced using PyMol with a recently determined crystal structure of YADH (PDB ID: 5ENV) to develop visual literacy skills • predicted the stereoselectivity of YADH with regards to the cofactor using the YADH structure • used a 1H NMR spectrum prediction program to understand how to experimentally discriminate between the pro-R and pro-S hydrogens on C4 of NADH.3,6,7 Prior to engaging in this activity, students in this course had completed training on using PyMol to visualize protein structures. A basic activity to introduce PyMol to students within the context of protein structural organization has been included (Supporting Information). During the second day or in-lab portion of this activity, students worked in pairs to mix reaction solutions with ethanol or ethanol-d6 and completed the 1 H NMR spectral analysis. During the final day or postlab portion, students connected the 1H NMR spectroscopy results to the stereoselectivity prediction from the YADH structure while more critically engaging with the primary literature article that accompanies the YADH structure.3 Using the 1H NMR spectra predictor program on NADH, students expected the pro-S proton on C4 to have an asymmetrical doublet centered at 2.69 ppm and the pro-R proton to have an asymmetrical doublet centered at 2.90 ppm.7 Setting up a reaction sample with undeuterated ethanol resulted in a fully proton-containing NADH, which results in peaks for both the pro-S and pro-R protons in the subsequent 1H NMR spectroscopy analysis (Figure 1A). Experimentally, the pro-S proton on C4 of NADH has an asymmetrical doublet centered at 2.66 ppm, and the pro-R proton has an asymmetrical doublet centered at 2.79. When ethanol-d6 is used as a substrate, the proton originally present on the aromatic C4 of NAD+ (approximate chemical shift of 9.0 ppm) is shown to be the pro-S proton with a final chemical shift of 2.64 ppm (Figure

Scheme 1. YADH Catalyzes the Oxidation of Ethanol to Acetaldehyde Coupled to the Reduction of NAD+ to NADHa

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The addition of semicarbazide makes this reaction irreversible by forming an imine with acetaldehyde in a non-enzymatic reaction.

NaOH, and 1371 U/mL YADH were prepared in D2O. Stock solutions were stored at −20 °C with the exception of NAD+, which must be prepared fresh due to proton exchange with the D2O solvent. Stock solutions were used to prepare 1 mL reaction samples with either ethanol-d6 or undeuterated ethanol in 5 mm glass NMR tubes. Reactions in D2O solvent contained 75 mM potassium pyrophosphate, 75 mM semicarbazide, 33 mM NaOH, 16 mM NAD+, 34 U of YADH, and 24 mM ethanol-d6 or ethanol. The reaction mixtures were secured within the NMR tube with a plastic cap, mixed well by inverting the NMR tube, and incubated at room temperature (approximately 21 °C) for 30 min. 1H NMR spectra of samples were obtained at 300 MHz.

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HAZARDS Semicarbazide hydrochloride is toxic. Sodium hydroxide solutions can cause severe skin burns. Ethanol is a flammable solvent. Stock solutions should be prepared by the instructor. Preparing and handling stock solutions and samples should be done with safety goggles and gloves. Stock solutions and samples should be disposed of in the appropriate waste container. B

DOI: 10.1021/acs.jchemed.6b00772 J. Chem. Educ. XXXX, XXX, XXX−XXX

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adds a hydride to the re face of the aromatic nicotinamide moiety of NAD+ to result in the final pro-R positioning of the transferred proton. To assess the success of this activity in terms of learning goals, brief pre- and post-attitudinal surveys and a postactivity content quiz were administered to students in the workshopformat course. For the attitudinal survey, students were asked to self-report on five questions (Figure 2). Four of the five questions asked about chirality, enzyme stereoselectivity, using 1 H NMR spectroscopy in biochemistry, and using 1H NMR spectroscopy to investigate chirality. Students reported gains in all four areas as evidenced by an increase in the median survey response. A fifth question about ability to use the 1H NMR instrument served as an internal negative control. Students in this upper-division biochemistry course have already completed a full-year sequence in organic chemistry during which competency in operating the 1H NMR instrument is developed. No gain in this area was observed or expected. On the postactivity content quiz, 72% of students were able to distinguish between pro-R and pro-S hydrides and identify re and si faces on chemical structures, whereas 28% of students were able to only identify re and si faces on a chemical structure and 6% of students were unable to distinguish prochiral hydrides or re and si faces. The activity presented herein was successful in allowing students to investigate the interactions between an enzyme (YADH), substrate, and cofactor. In addition, students used visual cues from a molecular structure of YADH and 1H NMR spectra to make and test predictions about the stereospecificity of the catalyzed reaction.

Figure 1. 1H NMR spectra of NADH produced during the oxidation of ethanol (A) or ethanol-d6 (B) by YADH. The pro-S and pro-R protons have asymmetric doublets with separated chemical shift values centered at 2.66 and 2.79 ppm, respectively (A). The absence of a peak in the chemical shift range from 2.725 to 2.85 ppm indicates that YADH transfers a deuteron from ethanol-d6 to the pro-R position of NADH (B).

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1B). The deuteron transferred from C1 of ethanol-d6 to the pro-R position on C4 of NADH does not result in a signal in the 1H NMR spectrum. The pro-R deuteron is inactive in geminal coupling with the pro-S proton that results in a single peak for the pro-S proton. The presence of a peak at 2.64 ppm indicates that (i) NADH was produced by the reaction because the proton peak shifts from 9.0 ppm and (ii) YADH selectively

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00772. Student handout with prelab, lab, and postlab activities; teacher handout with suggested answers to pre- and

Figure 2. Results of pre- and postsurvey questions assessing student attitudes. Increases in the median rating were found in comfort with chirality and enzyme stereoselectivity, use of 1H NMR spectroscopy in biochemistry, and use of 1H NMR spectroscopy to investigate chirality. A small increase in rating was found in the ability to use a 1H NMR instrument. Students in this upper-division course had already completed a year-long sequence in organic chemistry and developed competency in running a 1H NMR instrument. C

DOI: 10.1021/acs.jchemed.6b00772 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

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postlab questions and detailed instruction for solution preparation (PDF, DOCX) basic activity to introduce PyMol to students(PDF, DOCX)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Theodore J. Gries: 0000-0001-8942-6056 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Beloit College McNair Scholars program, the W.M. Keck Foundation, the Mead Witter Foundation, and the Mazur Family and Donald A. Anderson Science Funds for funding and to the students in the summer 2016 Independent Biology Research course and the fall 2016 DNA and Protein Biochemistry course.

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REFERENCES

(1) Oppenheimer, N. J.; Arnold, L. J.; Kaplan, N. O. A Structure of Pyridine Nucleotides in Solution. Proc. Natl. Acad. Sci. U. S. A. 1971, 68 (12), 3200−3205. (2) Mostad, S. B.; Glasfeld, A. Using high field NMR to determine dehydrogenase stereospecificity with respect to NADH: An undergraduate biochemistry lab. J. Chem. Educ. 1993, 70 (6), 504−506. (3) Plapp, B. V.; Charlier, H. A., Jr.; Ramaswamy, S. Mechanistic Implications from Structures of Yeast Alcohol Dehydrogenase Complexed with Coenzyme and an Alcohol. Arch. Biochem. Biophys. 2016, 591, 35−42. (4) Voet, D.; Voet, J. G. Biochemistry, 4th ed.; Wiley: Hoboken, NJ, 2010; pp 470−472. (5) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 5th ed.; W.H. Freeman: New York, 2008; pp 512−514. (6) The PyMOL Molecular Graphics System, version 1.8; Schrödinger, LLC. (7) Banfi, D.; Patiny, L. Resurrecting and processing NMR spectra on-line. Chimia 2008, 62 (4), 280−281 www.nmrdb.org.

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DOI: 10.1021/acs.jchemed.6b00772 J. Chem. Educ. XXXX, XXX, XXX−XXX