The Hydrolysis of pNitrophenyl Acetate: A Versatile Reaction To Study Enzyme Kinetics J. Anderson, T. Byrne, K. J. Woelfel, and J. E. ~ e a n ~ ' Seanle University, Seattle, WA 98122 G. T. Spyridis and Y. ~ o c k e r ' Department of Chemistry, BG 10, University of Washington, Seattle, WA 98195
A Versatile Mechanistic Probe for Three Enzymes I n a n earlier Dublication ( l a ) we described an e x ~ e r i ment involving ;he enzymatically catalyed hydrolyk of D-nitro~hrnvl acetate IPNPAI by bovine carbonic unhv-
cH3c02~o-No2
+. H
enz
CH3C02 + - 0 Since that time, many student projects have been derived from these experiments a t Seattle University and the University of Washington. They have allowed this part of our biochemistry laboratory activities to be expanded to include enzyme assays for two additional enzymes that also exhibit esterase activity The reaction of eq 1 is versatile: It is catalyzed not only by carbonic anhydrase but also by a-chymotrypsin (a-CT) and acetylcholinesterase (AcCE). Thus, a single reaction, convenientlymonitored, serves as a pedagogic tool and a mechanistic probe for three different enzymes. Like BCA (I), the latter two enzymes are commercially available, and their chilled solutions are stable for many hours or even days (2). We have developed simple kinetic assay methods to determine the specific and molecular activities of each of these enzymes by monitoring the formation ofp-nitrophenoxide (PNP) a t 410 nm. The assay procedures can be adapted to allow spectrophotometric determinations to be made using instruments as simple a s the Spectronic 20. Thus, a single reaction provides students with numerous projects that exemplify the use of kinetic studies as tools for the elucidation of the mechanisms of action for three enzymes. Physiological Function of the Enzymes BCAis a zinc metalloenzyme with a molecular weight of 30,000. Although it is a very versatile enzyme ( l b 4 , its physiological function lies in its catalysis of the reversible hydration of C02(la). By comparison, AcCE is a relatively large enzyme (molecular weight about 260,000) and functions a s a tetramer, each subunit having an active site (3). This enzyme plays a vital role i n the transmission of chemical signals between neurons by hydrolyzing the neurotransmitter, acetylcholine, at its postsynaptic receptor. AcCE CH3C02CH2CH2N(CH&+ H,O -+ ,
' Authors to whom correspondence should be addressed
To ensure that postsynaptic membranes return rapidly to a resting state, AcCE possesses the remarkable capability of turning over its substrate near the diffusion-controlled limit for a second-order rate constant. Under optimal conditions of pH, the value of k,,JK,,, is 1.6 x 10' M-' s-' (4). The nerve gases, such as diisopropylfluorophosphate, are potent inhibitors of AcCE due to their irreversible binding to the serine residue in the active catalytic site. The other subsite is negatively charged and forms salt bridges with acetylcholine in the enzyme-substrate complex. a-CT is a relatively small enzyme of molecular weight 24.800 (5a). The ~hvsiolo~cal . . .. role of the enzvme is the catalvsiiof h e hydrolysis of proteins. Caudysis om& most strowiy on the carbowlate side of hvdrophobir amino acids. ' h i c a l of the serine pr&eases, the active site region uses aspa& acid, histidine, and serine residues. In a-CT, there also exists a wide pccket lined with hydrophobic residues to accommodate hydrophobic amino acid protein-cleavage sites. Like AcCE, a-CT is irreversibly inhibited by.diisopropy1fluorophosphate. It is also irreversibly inhibited by N-tosyl-~phenylalaninechloromethyl ketone (TPCK), which tightly binds to the imidazole group of the histidine at the active site. Catalytic Versatility of a-CT The catalytic versatility of a-CT is such that i t also catalyzes the hydrolysis of p-nitrophenyl esters (5b). For experimental reasons, the hydrolyses of these esters have proven to be convenient reactions for the investigation of this enzyme. One of the interesting features of these reactions, when carried out using greater substrate than enzyme concentrations, is that the rate of appearance of PNP is biphasic (5).There is an initial and rapid liberation of PNP a t a concentration approximately equal to that of the enzyme, followed by a much slower zero-order release of PNP. This observation has been interpreted to be the result of the initial rapid formation of an acyl-enzyme intermediate and simultaneous release of stoichiometric amounts of the PNP leaving group.
Thus, the magnitude of the initial burst of PNP formation can be used as a n active-site titration for a-CT. ARer the initial burst, steady-state conditions are established, and the slower zerodrder hydrolysis ofthe acyl-enzyme intermediate releases free enzyme to continue the enzymatic reaction. Volume 71 Number 8 August 1994
715
photometric instrumentation does not have a thermostated cell compartment, all solutions should be preincubated at room temperature or in a constant-temperature bath regulated to 25 "C before kinetic runs. Experimental The enzyme assay experiment described below was designed to be completed in one laboratory period. At Seattle University, students have worked in pairs. The class may be split into three groups, each group carrying out the assay using one ofthe three enzymes. Because the same buffers, substrate solutions, and instruments are used by each group, the preparation and supervision of the laboratory for the instructor is greatly simplified. We also report in this paper some of our comparative findings of various kinetic parameters for the three enzvmes usine the substrate PNPA. We believe that these data will he'ip laboratory instructors in assisting students to develop projects for additional study Materials and Methods
Simple spectrophotometers, such as the Spectronic 20 (Milton Rov) are satisfacton, for this exoeriment. Recentlv one of our &structors and o& electronic>technician linked Spectronic 20 instruments to strip chart recorders (Kipp and Zonen, Bd 40) by means of an antilog amplifier that converts transmittance to absorbance. These inexpensive recording spectrophotometers allow students to use higher concentrations of enzymes. Also, due to the more rapid reaction rates that result., thev" can comolete the families of kinetic runs in shorter periods of time. In addition, we currently use Shimadzu 160U spectrophotometers interfaced with IBM compatible PC's that allow the students to manioulate collected data and carrv out substrate-versatilitv &dies that necessitate the ~ ~ ' V w c e . The ex~erimentalmethods described helow are mven assuming 5 . 0 - m ~cuvettes. When using spectrophotbmeters with 1-or 3-mL cuvettes, the volumes of all reaction solutions must be reduced proportionately, Disposable semimicro cuvettes may also he used in the latter cuvette holders. Because volumes of only 1mL are required for these cuvettes, this greatly reduces the quantities of the enzymes needed for the assay procedures. If the spectro-
Stock Solutions 1. 0.04 M phosphate buffer pH 7.4 2. 1.00 x lo4 M p-nitrophenol (Sigma) in 0.04 M phosphate buffer pH 7.4 3. (a) BCAfrom bovine erythrocytes (Sigma): 1.0 x 104M in 0.04 M phosphate buffer pH 7.4 (300 mglyaphilized BCA dissolved in 100 mL buffer) (b) a-CT (type I1 Sigma): 1.2 x lo4 M (by mass; see discussion) in 0.04 M phosphate buffer pH 7.4 (300 mg lyophilized enzyme dissolved in 100 mL buffer) (c) AcCE (type 111 Sigma, crystalline suspension or type M (depending on VS or VIS, lyophilized): about 4 x purity) in 0.04 M phosphate buffer pH 7.4 (5000 units crystalline suspension or lyophilized enzyme dissolved in 50 mL buffer) 4. 1.00 x lo-' M PNPA (Sigma) in l,2-dimethoxyethane or acetonitrile. We have generallv used the commercial moduct direetlv. ~owever.ifthe eim~oundamears vell&ish. it may be kclystalli~edfrom dikthyl ether to mnstant m.0. of 79-80 'C. -~ Caution:Dimethoxyetbane is flammable. On long exposure to light and oxygen it may farm ezplosiue peroxides (6).If disposable euvettes are used in the experiment, dimethoxyethane may not be used because it rapidly dissolves thepolymeric materials.
..
.~ ~~
~
a
~~
Experimental Protowl and Calculations
Standard Calibration Curve The extinction coescient of the ~-nitroohenoI&'Np is determined by taking absorbance r e a h g s o?various dilutions of solution 2 above with the ohomhate buffer (solution 1). These solutions should inclde lb% dimethoGethane because the reaction solutions will incorporate the same quantity of the solvent as used to introduce the PNF'Asubstrate. Kinetic Assay Procedure and Determination of SpecificActivity Students may begin with a kinetic run using 0.5 mL of the enzyme solution to be tested and 4 mL of buffer (solution 1).They may initiate the kinetic run by stiningin 0.5 mL of PNF'A in dimethoxyethane (solution 4). If the students are using manually operated spectrophotometers, an increase in absorbance of approximately 0.5 A in 5 or 10 min can be conveniently followed. If the reaction rates are more rapid, they may dilute the stock solution (solution 3) with a known volume of phosphate buffer to slow the rate accordingly The a-CT (3b) and AcCE solutions (3c) should not require dilution. Calculations of Results
Initial reaction velocities are calculated from plots of absorbance vs. time.
[Enzl X 10" (MI Figure 1. The hydrolysis of PNPAcatalyzed by (A) BCA A, AcCE 0 (8) a-CT 0.Student data determined at 25 "C, pH 7.4. 0.0 M phosphate buffersusing Spectronic 20 instruments. Molecular activities may be calculated from the slopes of the respective lines. The AcCE stock solutions used in the above knetic runs were about twice that given forsolution 3c. 716
Journal of Chemical Education
Molecular activities for the three enzymes are determined from the slopes of the lines obtained from plots of uinitial VS. the concentration of the enzyme (see, for example, Fig. 1).
F gLre 2. Determ nation of pH-rateprotlesof enzymes. (A) PNP enlnnion coenicenr as a f,nn!on of pH. 16)vbLno as a Lncr on of pH; (C, v,,, as a fmnion of OH for BCA. St~dentdata oeterm ned using a Spectronlc 20 mstrument Enzymar c rates were oetermned a1 25 'C in 0.04 M phosphate buffeis at [PNPA] = 1 x lo3 M.
Discussion
In our experimental protocol, we chose to use phosphate buffers to determine kinetic parameters for the three enzymes around neutrality To expand pH-rate profiles beyond the range of pH &8, we recommend using the combination of 2[N-morpholinolethanermlfonicacid, pK. = 6.1, and N-[2-hydroxyethyllpiperazine-N'-[2-ethanesdhkacid], pK. = 7.5. We usually use acetonitrile to dissolve the substrate. However, it causes partial inhibition of BCA(lb1, so it may be desirable to use 1,2-dimethoxyethaneas solvent. When certain lyophilized enzymes are dissolved in buffers a finite period of time is required to develop full enzymatic activity. For BCA, a typical period of about 1 h is usually satisfactory Thus, the solutions of the enzymes used in this experiment should be prepared about 2h before use and stored on ice or refrigerated. Typical determinations of molecular activity for BCA, a-CT, and AcCE are shown in Figure 1.At pH 7.4 in 0.04M phosphate buffer at 25 "C the relative activities are approximately 180 for BCA, 70 for AcCE, and 1for a-CT. Students will find that the absorbance vs. time plots for the hydrolysis of PNPAin the presence of a-CT do not extrapolate to zero absorbance. Again, this observation is consistent with the rapid formation of an acyl-enzyme intermediate and simultaneous release of stoichiometrie amounts of PNP (5).For example, in a typical kinetic run at pH 7.4 using an enzyme concentration of 1.1x 10" M, the extrapolated absorbance intercept was 0.103 (rather than 0.000). This would suggest that the actual concentration of active a-CT was AAkPNp = 0.103/15,000 M-' em-' = 6.3 x M. Thus, one can estimate that the sample of u-CT uscd in our experiments was approximately 11.1 x 10 5M1(6.3x 10-"MI&1003=63"r pure.NeitherBCAnor AcCE demonstrate the kinetic burst. Reaction Rates as a Function ofpH 'lo calculate reactlon rates as a function of pH, one must first determine the extinction coefficients at various values of pH in the range under consideration. Determinations such as those shown in Figure 2A allow the calculation of
the extinction coefficient based on the total concentration PNP @-nitrophenoxide + p-nitrophenol). Students will note that the point of inflection shown in Figure 2A corresponds to the known pK, of p-nitrophenol. Figure 2B represents the buffer-catalyzed rates of hydrolysis of PNPAin 0.04 M phosphate buffers. By determining total reaction velocities for the hydrolysis of PNPAin the presence of each of the three enzymes in phosphate buffers, the enzymatic components u,, may be calculated by subtracting the points in Figure 2B from the total rates determined at a given pH.
Molecular Activities at a Given p H Values of molecular activities a t a given pH may be calculated bv dividine .. u....... bv" the concentration of enzvme used in the experiment. Vpical student results arc shown for l3CAin Fieure 2C. The Dolnt of inflection for the same reaction cataGzed by a-CTis similar to that determined for BCA. The ocmrrence of these inflection points around neutrality is consistent with the known catalytic involvement of an imidazole residue functioningas a general base for both enzymes. On the other hand, students will find that the pH-rate profde for the same reaction catalyzed by AcCE is spread out over several units of pH. In this respect it is similar to that for the correspondinghydrolysis of acetylcholine. This similarity may indicate that certain mechanistic parallels exist between the latter reaction and the enzymatic hydrolysis of PNPA. ~~~
Kinetic Studies lb Demonstrate Mechanistic Properties In fact the data included in Figures 1 and 2 should enable laboratory instructors to help students develop and carry out many interesting kinetic studies that demonstrate important mechanistic properties of BCA, a-CT, and AcCE. At Seattle University, five periods of the first quarter of biochemistry laboratory are allocated for students to carry out projects oftheir choice. Although the instructor is available for consultation, each student pair has the responsibility Volume 71 Number 8 August 1994
717
.. .
to locate appropriate background articles i n the literature to outline their own experiments to prepare all solution^, including buffers to acquire, analyze, and interpret kinetic data
Upon completion of the project each student prepares a journal-style research paper and gives a short presentation to the class. Typical projects have included 1. The determination of K... and V-... as a function of pH 2. The determination of solvent dcutrrium imtope effects trsscntinlly pmjwt 1 abovr repcsted in DzOI' 3. The determination of inhlbrtrvn wnstantr K, far vanuus inhibitors of each of the three enzymes. (Examples could include sulfanamides and various anions for BCA (lb), nicotine, caffeine, and perhaps wmponents of commercial ehalinereie inhibitors found i n commerciallp available pest~cides(care in handling' for AcCE. Studlesun the kinetic irrewrsible~nhihitionuaingsctrvenite.drrected rengcnts (eg., TCPK for a-CT should be ~ n formative i n defining aetive-site structure.) 4. Quantitative analysis of the burst-phase kinetics for a-CT allowing the determination of the absolute concentration of the enzyme. Students may determine the level of purity of their a-CT samples by the appropriate analysis of linear plats of the concentration of a-CT calculated fmm the burst phase vs. the concentration of a-CT by mass. 5. Substrate versatility studies involving the determination and comparison of Michaelis parameters for other p-nitrophenyl esters. For a-CT see, far example, ref 70, and for BCA, ref 76. BCAalso catalyzes the reversible hydrations of aliphatic and heteraaramatie aldehydes (gal, alkyl pyruvate esters (861, pyruvate (8c), and glyoxylate (8d). ~
~
~~~~~~~~
~
~
Values of pD may be determined using a glass electrode as follows: pD = pH,ediy + 0.41.
718
Journal of Chemical Education
Several uf these latter rrnerions must he monitored in the W rrcgion, and due to them relatively rapid mtes, l h c ~ require recording speetrophotometers
Acknowledgment
We thank the National Science Foundation (Grant USE9152220) for providing S.U. the funds for spedrophotometric instrumentation. Special thanks to T.Griffith, Professor of Chemistry, and J. Trimble, Electronic Technician, who interfaced strip chart recorders to our Spectronic 20 spedmphotometers. We also thank S.U. biochemistry students Jamie David and Linda Widing who provided the data used in Figure 2. Finally, JEM thanks the S.U. Faculty Developement Committee for their support. YP thanks the Helen Bader Charitable Trusts and the Muscular Dystrophy Association for their partial support. Literature Cited 1. IalSpyridls, G.T.;Meany, J. E.: Poeker,Y. J. Chem. Edue. 1885.62, 1121. (blPaeker, Y.;Stane. J. T. Biochsmisw 1987.6.668: 1968,7,2936,3021,4139.(c1 Pocker, Y.; Janjie, N. J Blol Chom. 1388.263, 616. (d)For an
