Nucleophilic and Enzymic Catalysis of p~itrophenylacetate Hydrolysis Michael B. Head, Kalpna S. Mistry, Bernard J. Ridings, and Christopher A. smith1 Department of Biological Sciences, Manchester, M1 5GD, U.K. Mark J. Parker Department of Chemistry, The Manchester Metropolitan University, Manchester, MI 5GD, U.K Undergraduate studies i n biochemistry and chemistry usually emphasize the effectiveness of enzymes as catalytic agents, especially when compared with inorganic catalysts. However, first-degree biochemistry practicals involving enzymes are generally restricted to rather crude purification procedures or to relatively simple kinetic studies such a s estimations of the maximum initial velocity (V,,,) and the Michaelis-Menten constant (K,) and simple investigations of inhibition ( 1 3 ) . The most widely used measure of catalytic effectiveness, the specificity constant, k,.,lK, (MI,i s rarely determined presumably because in most cases t h e "real" concentration of enzyme i s not known. This article describes a simple procedure for determining the relative effectiveness of a number of amino acids and enzymes i n catalyzing the hydrolysis ofp-nitrophenylacetate. p-nitrophenylacetate+ H20 +p-nitrophenol + acetic acid
legend Table 11, and the complete experiment, including a 45-60-min incubation, may be completed in about 2 h. We give a careful and quite detailed introductory talk to the students before their experimental work. Students are advised (as always!) to think carefully about what they are doing and why they are doing it. I n particular, they are asked to consider what constitutes or defines catalysis in this specific case, to consider the structure of p-nitrophenylacetate, to read about the mechanism of catalysis by chymotrypsin (in, for example, Stryer (7), although most degree-level biochemistry textbooks h a v e s i m i l a r descriptions, or in references (8, 911, and to try and devise a general mechanism for the catalyzed hydrolysis. Clear descriptions of t h e common mechanisms of catalysis shown by enzymes are given in references (ll,12). The students usually arrive a t a mechanism based on that of chymotrypsin, in which the catalyst participates i n a nucleophilic attack on the carbonyl carbon.
Table 1. Typical Time-progress Data Showing the Changes in Absorbance at 405 nm Procedure Associated the Hydrolysis of pNitrophenylacetate by a Number of Catalysts This ester is an "artificial" substrate for a number of enzymes. It was used extensively in elucidating the mechanism of cataly0 5 10 15 20 25 35 45 60 sis of chymotrypsin (7-9) and is useful i n enzyme-based under- Control 0.221 0.231 0.300 0.135 0.153 0.100 0.120 0.035 0.078 graduate practical classes (e.g., 0.278 0.361 0.442 0.564 0.194 0.238 0.111 0.152 0.068 10). p-Nitrophenol i s almost Histidine white. However, a t alkaline pH i t lmidazole 0.651 0.741 0.915 1.078 1.308 0.459 0.556 0.359 0.255 exists as a n anion, p-nitrophen0.500 0.562 0.679 0.786 0.943 0.379 0.441 0.319 0.256 ate, which is yellow. Hence the Cysteine progress of hydrolysis i s easy to Serine 0.193 0.232 0.271 0.347 0.423 0.536 0.116 0.154 0.080 follow by determining the absor0.425 0.521 0.573 0.709 0.804 0.980 0.345 0.385 Chymotrypsin 0.259 bance a t 405 nm. We have used this simple pro- Lipase 0.423 0.495 0.539 0.736 0.740 0.890 0.348 0.387 0.265 cedure over the last eight years 0.785 0.898 1.136 1.288 1.510 0.514 0.594 0.415 with students attending basic Ficin 0.287 biochemistry and molecular biol%is siudent data was obtained by adding 1.0 mL of saturated pnitrophenylacetata (given as 8.3x lo4 M for an ogy units as part of their studies aqueous solution in 13) to a mixlure of 1.0 mL of stock solutions of each respective catalyst and 2.0 rnL of 0.05 M for a B. Sc. (Hons) degree in Ap- phosphate buffer, pH 7.0. Stack solutions of catalysts were imidazole, 0.5 mM; histidine, serine, and cysteine. 2.0 mM: plied Biological Sciences. These lipase and chyrnotrypsin. 5 mg1100 mL; and ficin, 0.5gl100 mL. All reagents were purchased from Sigma. Poole. Dorset, s t u d e n t s ' m a i n academic U. K. strengths a r e i n the biological sciences. The experiment h a s catalyst + p-nitrophenylaeetate 7utility in emphasizing a number of fundamental chemical catalyseacetate +p-nitrophenate features. The students are required to measure time-proThis is then followed by the release of the second product. gress curves for the hydrolysis of p-nitrophenylacetate in the presence of potential catalysts, which comprise several eatalyst-acetate + H20+catalyst + acetate amino acids and enzymes (Table 1). The rates ofhydrolysis in the presence of potential catalysts and i n their absence An understanding of these points also reinforces the idea (i.e., the control, 0.05 M phosphate buffer, pH 7.0) are then of polarity and the notion of polar compounds as having an unequal distribution of electrons. compared. The experimental procedures are simple (see Students are also requested to consider what constitutes 'To whom all correspondence should be addressed. relative catalytic efficiency, especially because the poten184
Journal of Chemical Education
Table 2. Summary of Catalysis of ~NitrophenylacetateUslng the Data Given in Table 1 and Partially Illustrated in the figure
Catalyst
Concentration in assay (M)
Rat? (min- )a
p~~~
k x lo4 (rnin" M-I)
0.0042
-
-
0.0083
6.04
0.0176
7.1
0.0115
8.5
controld
lo4
Histidine
5.0 x
lmidazole
lo4 5.0 x lo4 5.0 x lo4
Cysteine
1.25 x
3.95 51.7 7.04
Wk, x lo4
1 .O
1.95 25.5 3.48
of the catalytic,or nueleophilic, efficiency of each nucleophile is therefore given by Rate the ratio kJklk,. enhancement Nucleophilic attack by "simple" (mind mor') nucleophiles is generally relatively slow compared to the subsequent steps of the reaction and therefore is rate-limiting. How2.0s 26.8 ever, in the case of enzyme-catalyzed hydrolysis, both the forma3.65 tion of the enzyme-substrate A,~~SU,
complex and i t s subsequent breakdown into p-nitrophenate 0.0120 3348 and acyl-enzyme intermediate Chyrnotlypsine 5.8 x lo-' are relatively fast compared with Lipasee 2.5 x lo-' 0.0106 6400 the release of free enzyme and 4.8 x 10" 0.0211 88.0 acetate from the intermediate. Ficine This latter step is rate-detennin'Rate is defined as the change in absolbance with time. the case of amino acids,the DIGvalue is that of the side chain:. the .DIG-values for the .onmaw. calboxvl and amino in€! for the enzyme-catalyzed regmups of amino acids are 3&.3'and 6.8-7.9. action and makes the enzyme %alculated using the program listed in the Appendix fmm the correspondingsteady-staterates and concentrati0:is available for further acetylation of catalysts and assuming a concentration of 8.3 x loa M for pnifmphenylacetate (13. and production of p-nitrophen*0.05M phosphate buffer. pH 7.0. 'Assuming relative molecular masses of21,5M),SO,WO, and 26,000torchymotrypsin,lipase,and ficlnrespectively (14). ate' Thus, in the presence of excess substrate the reaction proceeds with a rapid initial burst phase followed by a slower "steady-state" (10). tial catalysts are used a t different concentrations. Their Because nucleophilic and enzyme catalysts act differlaboratory reports must include a definition of catalysis ently, it is clearly not straightforward to compare their and a discussion of the relative effectiveness of the amino catalytic mechanisms. It may he thought that the nucleoacids and enzymes as catalysts. philic ability of imidazole and the amino acids might be Typical results for this experiment are given in Tables 1 closely related to their basicities (pK, values). This is and 2 and the figure. clearly not the case with this experiment (Table 2) and, inAnalysis of Results deed, nucleophilicity-basicity correlations occur only under certain narrow limitations (15).However, under the Catalysis can be demonstrated to have occurred when conditions of the assay, a simple general measure of catathe rate of hydrolysis is greater in the presence of the polytic effectiveness can be given as a molar rate enhancetential catalyst than in its absence, that is, in thecontrol. ment, defined as This is the case for all the amino acids and enzymes listed in Table 2. However, these are operating a t different molar 'a%td,ed - rateCo"*,l concentrations, and this must be taken into account when amount of catalyst considering. their relative effectivenessas catalysts. The catzyzed hydrolysis ofp-nitrophenylacetate by conwhich, in this experiment, will have units of min-'mol-I. ventional nucleonhiles is essentiallv a nucleo~hilicacvl substitution reaciion with an initial attack on the carbonil Conclusion carbon by the nucleophilic species and the subsequent hyThis simple, reliable practical introduces and reinforces drolysis of the intermediate. In the absence of a catalytic a nUmber ofhasic chemical points including species, Serine
0.0076
13.6
3.28
1.62
1.70
rate of reaction = k,[substratel where k, is the observed rate constant in the presence of phosphate buffer and
However, in the presence of an added nucleophile (Nu), the corresponding rate equation is rate of readion = k&ubstratel+ k,[Nul[substratel where k, is the rate constant describing the nncleophilic attack by the added nucleophile. Rearrangement of the above equations yields the following expression. k, =
(rate - kJsubstrate1) [Nu] [substratel
I I ~stralesselecteo oata from Table 1 Tme-progress cJwes are gven for the control 0 05 M phospnale ouffer,pH 7 0 (L),h sl o ne (A), chymotlypsn (W), and im dazole (T)
Volume 72 Number 2 February 1995
185
Appendix nn5 ""- c1.s 010 PRINT "THIS PROGRAM OBTAINS RATE CONSTANTS FOR BOTH THE HYDROXL-CATALYZED HYDROLYSIS OF A SUBSTRATE AND COMPARES IT TO THE HYDROLYSIS PERFORMED IN THE PRESENCE OF ANOTHER NUCLEOPHILE"
---
- --
"90 PRTNT .. . ... .":w*******************X***********'*******************~
030 INPUT 'Enter the concentrations of substrate and catalyst'; sb, ct 040pRINT"**:**************************************************,, 050 INPUT 'Enter the rate of reaction in control (min-'1"; a 1 o6opRINT"****************************************************" 070 INPUT "Enter the rate of the reaction in the presence of added catalyst (min-'1"; a2 80 pRINT"***"***********************%**I*********************,, 90 k , = a l / s b 100 kc= (a2 - (k. * sb)) 1(sb * ct) 110 PRINT "k, (hydroxyl catalyst) = "; k,;" min-' M-'"
120pRINT"**************************************************~
130 PRINT "k, (added nucleophile/catalystj = "; kc; "min-' M-'" 140 PRINT "************************:i;e************************" 150 r t = k , / k , 160 PRINT "relative efficiency of nucleophile = ";rt
I70pRTNT"****************************************************-7 180 INI'IJ'I' "ANOTHER CATALYS'I' YIN ? "; QS = " Y O R O$ = "v'"I'I1EN GOT0 30 190 -1k'OS ~
~~
~
~
200 E L < IF ~ &= ...V'' OR"Q$ = ..n" THFN GOT0 230 EI.Sk: I'RIKT "INCORRECT E.U'I'I
