Course development was supported by equipment funds from the Youngstown State University Dean's Advisory Council and in part by a grant (to J. A. S.) from the American Chemical Society Petroleum Research Fund.
The enzyme orotidine-5′-monophosphate decarboxylase is an attractive choice for the central theme of an integrated, research-based biochemistry laboratory course. A series of laboratory exercises common to most instructional laboratories, including enzyme assays, protein purification, enzymatic characterization, elementary kinetics, and recombinant DNA methods, has been devised. The series of exercises requires biochemical instrumentation common to most laboratories and some specialized materials available from the author's institution to laboratory instructors.
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This article is sincerely dedicated to the life and career of Dr. Mary Ellen Jones (1922–1996). See Ref. 1.
Biochemical educators are becoming increasingly aware that biochemistry laboratory courses are often disjointed, in that different experimental methods for protein and enzyme analysis are introduced using different proteins and enzymes . The disjointed nature of some laboratory courses is usually because of the fact that different commercially available (and affordable) enzymes and proteins are better suited than others for demonstrating a biochemical principle. However, a collection of laboratory exercises using many different proteins does not introduce students to the continuing nature of biochemical research. That is, the idea that the procedures performed a week ago may have significant bearing on the procedure of the current week is not always appreciated by undergraduate biochemistry laboratory students, yet this is almost always the case in biochemical research.
Youngstown State University, Youngstown, OH has initiated the biochemistry option for the Bachelor of Science degree in chemistry according to American Chemical Society guidelines. This degree curriculum includes a second semester of biochemistry laboratory and is intended to prepare students for undergraduate research in their third and/or fourth years. To meet these goals, our department has recently begun to offer a course entitled Enzyme Analysis. The course is structured to introduce students to intermediate biochemical laboratory techniques, using the same enzyme (and its gene) throughout the entire semester and thus more closely resembling the progress of a long term research undertaking.
The enzyme under study in this course is orotidine-5′-monophosphate decarboxylase (OMP decarboxylase, ODCase;11 see Fig. 1), an enzyme of great current interest among enzymologists [3–7] because of its enormous rate enhancement and incompletely characterized mechanism. This enzyme has many significant features that make it an attractive choice for an integrated, research-based laboratory course. 1) ODCase is part of the de novo pyrimidine biosynthetic pathway, a topic that is usually covered in metabolic biochemistry; 2) The absence of ODCase is related to the human hereditary metabolic deficiency orotic aciduria; 3) The ODCase gene, ura3, is a common marker in yeast genetics; 4) Over 65 sequences of ODCases from different organisms have been determined , and the isolation of an ODCase gene from gene libraries is relatively easy, using any of a number of ODCase-deficient bacterial strains; 5) ODCase has been found to have the largest rate enhancement, kcat/kuncat, of any known enzyme and has thus been dubbed the most proficient enzyme ; 6) Many recent reports on the chemical mechanism of ODCase have been published [10–33]. Different proposed mechanisms have support from various studies, and a healthy debate exists among ODCase aficionados. This can form the basis of a critical literature review student assignment; 7) Most importantly, ODCase is amenable to many useful biochemical laboratory exercises, introducing some intermediate biochemical concepts, as will be outlined herein. Most experiments are similar to those used in general biochemistry laboratories (spectrophotometric assays, gel electrophoresis, etc.) but are unified toward the common theme.
MATERIALS AND METHODS
Many materials and reagents for this laboratory course are commonly used in most biochemical teaching laboratories. These specialized materials are available from the following sources.
The Saccharomyces cerevisiae system capable of high level ODCase production was developed by Bell and Jones  and is still in use in our laboratory. We have obtained as much as 70 mg of purified ODCase from four liters of yeast liquid culture. This strain is available from our laboratory for educational and research purposes. A preparation of purified ODCase is needed for control trials in the experiments outlined. This can be accomplished in 5 or 6 days (3 days of yeast growth, a few days of protein purification), according to the Bell and Jones purification procedure . The purified protein obtained from the Affi-Gel blue column is sufficiently pure for all procedures herein. The enzyme is stable for several months when stored in 20% glycerol at −20 °C.
A small, convenient instrument for lysis of small volumes of yeast is necessary. We obtain good results using BeadBeater products from Biospec, Inc., Bartlesville, OK.
Affi-Gel blue affinity chromatography resin is available from Bio-Rad. 6-Azauridine-5′-monophosphate (6-azaUMP), a competitive inhibitor of ODCase used in affinity chromatography, has been obtained from RI Chemicals, Orange, CA.
OMP, the substrate for ODCase, is almost prohibitively expensive from Sigma. ($1156 per 100 mg). However, we have synthesized OMP in usable quantities from UMP ($58.80 for 10 g; Sigma) by adaptations of the method of Ueda et al. . The desired product can be separated from unreacted starting materials using anion-exchange chromatography.22 For departments with substantial laboratory budgets, the 100 mg of OMP from Sigma is enough for over 4600 spectrophotometric assays.
ODCase-deficient E. coli.
This strain (pyr4−) is needed for the exercise on selection of an ODCase gene from a gene library. Many such strains are available from various laboratories; ours is available for educational and research purposes.
Gene Libraries for ODCase Gene Screening
We have used the Neurospora crassa libraries from the Fungal Genetics Stock Center, Department of Microbiology, University of Kansas Medical School and obtained the ODCase gene first isolated by Newbury et al. . These should be used as the M13 form; λ-phage libraries can be converted to the M13 form by a mass excision protocol. Samples of the N. crassa gene library M13 phage form are available on a limited basis from our laboratory. Dozens of ODCase genes from various organisms have been identified by genetic complementation; many different gene libraries may be useful for this exercise.
Other necessary laboratory instrumentation is as follows: UV spectrophotmeters, microbial incubator-shaker for large cultures, incubator for agar plates, Sorvall refrigerated centrifuge or equivalent, microcentrifuges, agarose gel and polyacrylamide gel electrophoresis apparatuses, and an autoclave.
This laboratory course is offered independently of the second-semester biochemistry lecture course and is held for one 3-h laboratory period per week. A laboratory assistant is particularly helpful for sterilizing and inoculating cultures at designated times before the laboratory period.
Portions of Ref. 37 and Ref. 38 are used as supplements for the course. A collection of detailed laboratory exercises has been compiled and will be available for distribution in Spring 2002.
Week 1, Handouts on Micropipeters, Dilutions, Amino Acids, and Protein Structure
An exercise on protein determination via Bradford assay will be performed. Bradford protein assay reagent (Pierce) is supplied with bovine serum albumin standards. Standard curves are prepared, and the slope relating absorbance and μg of protein is determined using Microsoft Excel.
Points to Emphasize for Protein Concentration Determinations—
The standard curve for the Bradford assay is most easily used for determination of unknowns when μg of protein, rather than protein concentration, is plotted on the horizontal axis. The protein concentrations of the standards are usually constant before addition to the reagent; the volume added to different samples is variable. Students have been found to show some unnecessary confusion when protein amount on a standard curve is expressed as concentration.
Week 2, Spectrophotometric ODCase Assays
The standard ODCase assay in all exercises contains 50 μM OMP, 10 mM Tris-HCl, pH 7.4, and protein in 1.0-ml reaction mixtures. Typical stock solutions in our laboratory are composed of 500 μM OMP and 100 mM or 1 M Tris-HCl. Purified or partially purified ODCase is used; in subsequent experiments, students will use ODCase from their own preparations. Spectrophotometric assays can be initiated by addition of protein as the last component if the ODCase is concentrated and does not interfere with the absorbance of the OMP. Otherwise, assays are initiated by addition of OMP as the last component.
The spectrophotometric assay is used in many of the remaining exercises in the course, and rapid calculation of spectrophotometric data is essential. Students record UV spectra from 220 to 320 nm; the absorbance change for the ODCase reaction is measured at 286 nm, and the difference extinction coefficient (Δϵ286) is −2250 M−1 cm−1 .
Points to Emphasize for Spectrophotometric Assays—
ODCase can be assayed over a period of time for which the decrease in absorbance is linear. Absorbance change can easily be monitored every 10 s even without automated monitoring. The spectrophotometric assay is limited by concentration range; there must be enough substrate to detect with the spectrophotometer but not so much that the absorbances are greater than ∼0.8. This limits the utility of the assay in obtaining kinetic data (see Weeks 7 and 9).
Often, enzyme preparations are too concentrated for easy enzyme assays (the absorbance change is finished in a matter of seconds). It is a good exercise to provide students with a sample of protein and include in the assignment that they must add the right amount of protein (which may require a dilution) to obtain a substantial absorbance change within a reasonable period, usually 1 min.
Week 3, Induction of ODCase Activity by Addition of Galactose
The yeast system used in our laboratory produces elevated amounts of ODCase when induced with galactose . Students use the spectrophotometric assay to measure ODCase activity levels in cultures induced for varying amounts of incubation time or with variable amounts of added galactose. The Bradford assay is used to determine protein concentrations in the yeast lysates, allowing students to calculate specific activity in nmol min−1 μg−1. For this exercise, yeast cultures are started 2 days before the laboratory period and induced with galactose in the evening before the laboratory period.
Week 4, Partial Purification of ODCase Using Ammonium Sulfate Fractionation
ODCase can be partially purified using a 60–90% ammonium sulfate fractionation. The presence of the enzyme is quickly detected and quantitated using the spectrophotometric assay. The specific activity in the redissolved 90% pellet can be calculated using the protein concentration from the Bradford assay; the specific activity should be higher in the redissolved 90% pellet than in the original yeast lysate. For this exercise, yeast cultures are cultivated as for Week 3.
Week 5, Affinity Chromatography Using Affi-Gel blue 
For this exercise, yeast cultures (1 liter is necessary for six pairs of students; 2 liters are better) are cultivated and harvested and fractionated with ammonium sulfate on the day before the lab. The 90% ammonium sulfate pellet is redissolved in a minimal volume of buffer and dialyzed overnight. On the day of the lab period, students (usually working in pairs) are provided with a 1–2-ml sample of dialyzed protein. This sample is applied to a 2.5 × 5-cm column of Affi-Gel blue and eluted with buffer. Fractions are monitored for total protein by a qualitative Bradford test (students do not measure the absorbance; they simply look for the absence of a color change). Generally, about 20 fractions of 2–3 ml will be collected, whereupon the total protein concentration in the final fractions is undetectable. ODCase is fairly stable to this purification process carried out at room temperature; we used chilled buffers and transfer fractions to ice upon collection. When total protein eluting from the column is undetectable, the elution buffer is changed to include 50 μM 6-azaUMP. ODCase will elute quickly upon changing the buffer, usually within 8 to 10 fractions. Despite the presence of the competitive inhibitor 6-azaUMP, enzyme activity in the fractions can be detected by the spectrophotometric assay. Glycerol is added to each fraction to a final concentration of 20%, and the fractions are stored at −20 °C for subsequent use.
Week 6, Detection of Protein Purity by Polyacrylamide Gel Electrophoresis
Sample wells should be large enough to contain 20–30-μl samples. For ODCase (monomer molecular weight = 29,000) in 12% gels, the migration of bromphenol blue tracking dye to the bottom of the gel is coincident with migration of ODCase to slightly below the middle of the gel. Samples should include chromatography fractions from the previous week in which the presence of ODCase is suspected or confirmed by spectrophotometric assay and pre-purified ODCase standard. ODCase in these column fractions is detectable by Coomassie Blue staining.
Week 7, Measurement of Enzyme Activity under Non-optimal Conditions: pH Profile
ODCase is fully active in a wide variety of buffers (except phosphate, a mild competitive inhibitor). We have used acetate, MES, MOPS, and Tris to cover a pH range of 4.5 to 9.5. The variation of pH produces a typical activity curve for ODCase.
For this experiment, all students should use equal volumes of the same ODCase preparation for enzyme assays. If a student ODCase sample is to be used, it must be treated to remove 6-azaUMP. This can be accomplished by dialysis or Sephadex desalting.
The spectrophotometric ODCase assay uses an OMP concentration of 50 μM, well above the measured Km (0.7 μM for the yeast enzyme). Thus, spectrophotometric assays are carried out at essentially saturating (Vmax) conditions, and the pH dependence is a rough measure of the variation of Vmax with changing pH . The implications of the variations of Vmax or Vmax/Km can be a subject of a laboratory/lecture presentation. The variation of Vmax/Km with pH for ODCase has been measured using the radioactivity assay ; student results can be compared with these previous results.
Week 8, Measurement of Enzyme Activity under Non-optimal Conditions: Thermal Denaturation
Yeast ODCase begins to become inactivated upon 5-min exposures to temperatures of 45–65 °C . Remaining activity is measured using the spectrophotometric assay; results can be expressed as a fraction of unheated ODCase. Students who were unsuccessful in obtaining adequate ODCase protein from Week 7 can use unpurified enzyme.
Week 9, Measurement of Kinetic Values Km and Vmax
The discussion of enzyme kinetics in Week 7 should lead to the conclusion that Km cannot be measured with the spectrophotometric assay, because the necessary concentration of OMP is saturating. Km and Vmax have been measured using the 14CO2 displacement assay , which can be accommodated for a wider range of substrate concentrations. Because the 14C assay is impractical for undergraduate laboratories, we have implemented a computer laboratory session for manipulation of mock data.
In this exercise, students are first introduced to the concept of specific activity of a radiolabeled compound (as distinguished from the specific activity of an enzyme preparation). With cpm data, specific activity in cpm/nmol, assay times, and protein amount, students calculate enzyme activities and compare with the calculations from spectrophotometric data.
A Microsoft Excel program has been prepared to calculate enzyme velocities from arbitrary values of Km and Vmax (which can be selected to match those for ODCase). In this exercise, students enter their own substrate concentrations and obtain calculated velocities. These values are automatically calculated into 1/V and 1/[S] values and generated into a Lineweaver-Burk plot. Random fluctuation of the calculated velocities can be incorporated into the Excel program to simulate experimental error. Copies of this Excel document are available upon request.
Week 10, Measurement of Inhibition Constants for Inhibitors of Various Modes 
As is the case with Km, inhibition constants cannot be measured effectively using the spectrophotometric assay. A second Microsoft Excel spreadsheet has been prepared in which velocities are calculated for [S] and [I] values entered by the student. The spreadsheet program calculates velocities for competitive, non-competitive, and uncompetitive modes of inhibition at predetermined Km, Vmax, and Ki values.
In this exercise, students first obtain the Km and Vmax values for a hypothetical enzyme by entering [S] values iteratively until an acceptable Lineweaver-Burk plot ([S] is in the range of Km) is obtained. Then, to determine the Ki values, students set up sets of virtual reactions with [S] values near the Km and begin to enter [I] values until acceptable degrees of inhibition are obtained, as judged by the resulting kinetic plots (automatically generated and representative of the type of inhibition). Students manually obtain the values of the apparent changing kinetic constant (Km, Vmax, or both) and generate a replot to determine the value of Ki.
Week 11, Bacterial Complementation and Genetic Screening
The yeast plasmid pGU2, used for yeast overproduction of ODCase, contains the ampicillin-resistance gene and origin of replication for propagation in E. coli. Plasmid pGU2, when introduced into an ODCase-deficient E. coli strain, will allow growth on media containing no pyrimidine source. Uracil or uridine will supplement growth of our pyrimidine auxotroph. This exercise begins with the introduction of plasmid pGU2 into CaCl2-treated E. coli and plating on agar media with no pyrimidine source (composition of media is available on request).
In the second portion of this exercise, a cDNA library in the form of M13 phage is used to infect samples of the ODCase-deficient E. coli strain, and the infected cells are plated onto agar media with no pyrimidine source. Control samples are plated onto complete medium with ampicillin selection to determine the phage titer. Students (working in pairs) produce 10 plates at ∼2000 colonies per plate.
After 2–3 days of incubation, colonies will appear for bacteria infected with phage carrying the ODCase gene. We have obtained consistently positive results with our N. crassa cDNA library (success rate for obtaining the ODCase gene is about 50% of student pairs). With our E. coli strain, false positives are common. True positives can be verified by means of a plasmid miniprep from each cultivated colony and reintroduction of the isolated plasmid into fresh ODCase-deficient E. coli, whereupon only those cells transformed with an ODCase-encoding phagemid should grow abundantly on agar media with no pyrimidine source.
Week 12, Mutagenesis and Isolation of Non-functional ODCase Genes by Positive Selection
A non-functional ODCase gene can be identified by its inability to complete the pyrimidine metabolic pathway in the presence of 5-fluoro-orotate (5FOA) in the media. When ODCase is absent, 5-fluoro-OMP produced from 5FOA will not be decarboxylated and will not lead to formation of toxic fluorinated pyrimidines. Cells with no ODCase activity can be identified by their ability to grow on media containing 5FOA, with uracil or uridine as the pyrimidine source.
ODCase-containing plasmids from successful screening experiments (Week 11) can be subjected to treatment with a mutagenic source; we have used low wavelength UV light rather than chemical mutagens. The degree of mutagenesis can be assessed by the number of colonies arising on 5FOA media.
The Internet site ca.expasy.org/enzyme/ contains links to published information on most known enzymes. This site contains gene and protein sequence information for 65 ODCases (enter EC number 22.214.171.124 for quick access). From this site, protein sequence information for any number of ODCases can be copied and transferred to the sequence alignment program at searchlauncher.bcm.tmc.edu/. Active site amino acid residues for ODCases are well defined (Lys-59 through Asp-96 of the yeast enzyme), highly conserved across species, and easily identified upon comparison of two sequences. Bacterial ODCases have significant differences from their eukaryotic counterparts. The orotate phosphoribosyltransferase domain of the bifunctional UMP synthases of mammals is easily distinguishable from the ODCase domain.
The following are optional exercises as time and resources allow. 1) Dimer formation of yeast ODCase in the presence of ligands ; 2) Monitoring the ODCase reaction by 1H NMR. This assay utilizes the difference in the chemical shift of H5 in the substrate and product (5.6 ppm singlet in OMP; 5.8 ppm doublet coincident with the H1′ signal in UMP).
SAMPLE STUDENT DATA
Some student data for representative experiments, such as the ODCase pH profile (Fig. 2) and thermal denaturation (Fig. 3), can be compared with reports in the recent biochemical literature. As discussed in the Week 7 experiment, the spectrophotometric assay of ODCase must utilize a concentration of substrate OMP that is essentially saturating. Thus the variation in activity with changing pH is a measure of the titration of the ES complex. Fig. 2 shows a titration with pKa = 6.2. By contrast, the measurement of the pH-dependent variation of Vmax/Km , which requires the 14CO2 assay, indicates a titration of either free E or free S, and the corresponding value found for either free E or free S is closer to 7.0.
Fig. 3 shows the decreasing ODCase activity as it is exposed to higher denaturing temperatures. The shallower slopes for the enzyme assays with denatured enzyme can be converted to activity using the difference extinction coefficient for OMP to UMP; this calculation can then be compared with the thermal denaturation data recently published for the yeast enzyme . Note that this study compared the denaturation temperatures for recombinant yeast ODCase produced in yeast versus recombinant yeast ODCase produced in E. coli.
Connectivity of Experiments throughout the Course
Protein determinations and spectrophotometric ODCase assays, presented in the first weeks of the course, are utilized continuously in Weeks 3–8. In Weeks 9–10, the shift from continuous spectrophotometric assays where the concentrations of UMP are detected, to the (virtual) end point radioisotope assays where the amounts of 14CO2 are measured, should be clarified and emphasized. The ability of the enzyme to generate UMP in vitro, and thus complete the de novo pyrimidine biosynthetic pathway, and the necessity for providing preformed pyrimidines to ODCase mutant strains in the bacterial complementation experiments, is a connection between metabolic biochemistry and enzymology that is easily made.
Benefits and Drawbacks of ODCase as the Central Enzyme in an Integrated, Research-based Laboratory Course
The main drawback for using ODCase in this course has been the expense of, or effort necessary to, synthesize OMP. However, the compound can be synthesized by an undergraduate with experience in organic chemistry as a summer project. A rigorously pure preparation of OMP is not necessary for most experiments, because the synthetic intermediates are only weak ODCase inhibitors. The disadvantage of the spectrophotometric assay for kinetic determinations seems to have been successfully overcome by the computer simulation programs.
The main benefit of using this enzyme in this course has been the ease of interfacing biochemistry (protein and enzyme assays) with molecular biology (recombinant DNA technology). The switch from enzymology to gene manipulation and analysis in Week 11 may appear awkward but actually proceeds quite smoothly.
I thank the following Youngstown State University students who assisted in the development of this course while enrolled in the first offering of the course in Spring 2001: Maggie Braun, Anna Dashkevich, Brian DelFraino, Juliana Ginocchi, Kathryn Kitzmiller, Christine Novicky, Miltos Ntragatakis, Jada Reed, Jody Regula, Yuriko Root, Heather Trenary, and Jennifer Vodhanel. I am grateful for the collaborations with all of the ODCase aficionados worldwide (listed as authors in the cited references).
The abbreviations used are: ODCase, orotidine-5′-monophosphate decarboxylase; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; 5FOA, 5-fluoro-orotate; 6-azaUMP, 6-azauridine-5′-monophosphate.
Details of OMP synthesis and purification are available upon request.