A Dynamic Model to Demonstrate Enzyme Activity
Terry L. Helser S.U.N.Y. at Oneonta, NY 13820-4015 T h e bane, whoosh, and flash of introductory chemistry demonstraGonsl are almost absent from biochemistry lectures, a t least in m y experience. Since overhead projections andslides d o n o t seem t o have the i m o a d o f ademonstration or model, I have been trying to find o; develop useful ones for several vears. While accurate and dvnamic models for the DNA h e k and t h e ribosome have eluded m e t o date, I have develooed a rather effective model for a n enzvme and its active site from a foam football and readily available craft suoolies. It demonstrates "induced fit"of suhstrate into the ac&e site, withshape changes in both and bond strain in t h e substrate. It can also show competitive and noncompetitive inhibition.
Materials One (or more), two-colored foam football, Bunsen burner, thinbladed spatula, 32-mm-diameter (l%in.-for nitrogen) and 38-mm (I1/?-in.-for carbon) styrofoam balls, '12-in. white (hydrogen) and 1in. red (oxygen) "POMPOMS" (Fibre-Craft Materials Corp., Niles, IL 60648), 12.5-mm-diameter (%-in.) Velcro spot fasteners, black and blue spray paint, and model glue (The Testor Corp., Rockford, IL 61101). Dlrectlons Make the substrate first, and then use this to outline the active site on the football. I used a peptide hond as my substrate, with styrofoam halls for the carbon and nitrogen atoms and fiber halls ("porn poms") representing the oxygen and hydrogen atoms. The hond angles and covalent radii were derived from a model kit2using aO.1-nmto about 2-cm scale (see Fig. 1). The C-C and C-N bond positions on the styrofoam balls were flattened with sandpaper and by rotating them against each other prior to gluing to simulate electron shell overlap. The C-C and C-N pairs were then spray painted. When dry, the porn pams were just glued in position. The oartial double bond between the carbon and nitroeen was simulated hy indenting arirrular ares ahout I mm rntueachflattrned ballnnd gluing m e side of the iartener spot into each hall. This keeps the fastener from being visiblp and increasing the hond length. Since this is a hydrolysis, water can be made by gluing a white porn pom onto a red one, and at 104.5". gluing one side of a velcro spot. Glue thr other sideofthespot toanother white yum pom. Besurr rhnt the O-H fastener spot will stick to theC=Oaideaf your peptide hond and the H- fastener will stick to the N-H side! You will make a carboxyl and an amino group after hydrolysis. The foam can be cut easily and precisely using a heated blade or wire, hut this produces copious amounts of toxic fumes, so do this only with ample ventilation or in a hood. To start, use the heated spatula hlade (or hacksaw) tocut across in the middleofthe bottom (opposite the "Laces") about two-thirds of the depth to the center. The intersection of the cuts will coincide with the C=N hond to he hydrolyzed. Cut a wedge (the noncompetitive inhibitor), in the top of the ball, measuring about 6 cm along the "laces" and almost to the center of the ball. This wedge is from the short dimension of the ball
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' For example, see Rehfeld, D. W.; Barondeau. M. J. Chem. Educ. 1988,65894-895; Peyser, J. R.: Luoma. J. R. J. Chem Educ. 1988, 65.452: and Pran. G.: Curtright. R. D.: Hill. L.: Clark. S. J. Chem. Educ. 1988.65.896-897, Lowrie, R. S. Minit Molecular Building System; Cochranes of Oxford Ltd., Leafield, Oxford OX8 5NT, U.K., 1973. 286
Journal of Chemical Education
F~gure1 The substrate model A d agram of me atomic d mens on5 and bond eng es 01 me peptlds oono "sea as a s~oslraletor the faatoaI protease ' Tne tnree other gr0.p~ bonded lomea pha caroons are not mcl-ded for slmploc ry and clarity
(see Fig. 2). Paint this a different color than either half of the ball. With the first wedge removed, cut a second wedge along the longest dimension of the topof the ball, about 3-4 cm wideand toahout the center of the ball. Discard the wedge pieces removed. Compressing this wedge-shaped cavity together should open the longest cut of the cross on the other side of the hall. Compressing the shorter cavity shouldopen theshortercut. Insertingthepainted wedge initscavity should make either compression difficult. Lay the substrate so the -N=Cbond extends parallel to the longer cut of the cross on the ball. Compress the longest wedge on the other side to open the longest cut slightly, tape it to hold it this way, and trace the substrate's outline on the surface. Use the hot spatula (or a power drill rasp) to gouge out the foam to a depth of about 2 cm. The resulting pocket need not be precise or fit the substrate exactly, since real active sites are not "lock and key". To illustrate "induced fit", the oacket should he smaller than the substrate when the bail is relaxed and alhu thesuhatrate todrop int~,it when thc longer w d g e tavity is tully conlpre~~ed. ReIrase 01 the compreAon should grip the suhstrate in the pucket.and better yet, strain the -C=K- fastener. You may have to ruin a couple balls before you make one that is acceptable, particularly if you do not do multiple fittings as you remove foam. ~~
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Demonstrallons Posslble wllh the Model T h e different colors of the two sides of the ball can simulate t h e multiple subunits of regulatory enzymes or different domains in a single chain, as you wish. T h e painted wedge is a noncompetitive inhibitor. T h e reaction sequence shown in Figure 3 can b e demonstrated a s follows. With the wedge removed, place the substrate over t h e active site. Compress and release the loneest cavitv on t h e other side t o demonstrate substrate h i d i n g i n t o i h e active site and to simulate "induced fir" and the conformational strain induced in suhstrate and enzyme. Compress t h e short wedge cavity to rip t h e C=N fastener apart, simulating hydrolysis. Keeping
Figure 3. Tha reaction. The peptide bond model is shown (bollom) in the bonded, trans configuration, hydrolyzed to show the fasteners, and combined with water (top) to fwm the amino end of one peptide (left) and the carboxyl end of another aner hydrolysis. Eimer fragment at the top can be used to simulate a competitive inhibitor.
Figure 2. The enzymmodal. The foam fwtbali is shown (Za) with both bottom Cavities slightly compressed to open the two outs anass the active site. The "nonc~mpetitlveinhibitor" wedge must be out of its site todo this. The second panel (2b) shows the wedge inserted to prevent compression and merefore "actlviW'.
this cavity compressed and compressing the longer cavity as well should release the two fragments, possibly with a little shake and gravity assist. T o complete the hydrolysis, split the O-H and H- from a water molecule and attach the H- t o the C-N'-H to make the amino group, and the -0-H to the C-C'=O tomake the carhoxyl group. Witha little practice and three or four hands, you might even he able to do this while the two parts of the substrate are still in the active site (good luck!). Noncompetitive inhibition is simulated by inserting the
wedge in its cavity opposite the active site (see Fig. 2b). This should make i t difficult to insert the substrate or compress the ball to split the fastener representing the C=N bond. Competitive inhibition can he demonstrated by inserting a C-C02H or C-NH2 fragment into the active site and then trying to bind a complete peptide substrate as well. Although the relative dimensions of active site to football are undoubtedly too similar to be at all accurate for an enzyme, they still do give some impression of the small fraction of the structure of an enzyme devoted to the active site. Drawing attention to it will at least help eliminate a common student misconception that an enzyme is all active site. Finally, the flexibility and changing shapes required to demonstrate with this model do help to impress students that enzymes are not the static, flat things they see in textbooks, but are dynamic, 3-dimensional structures. And, if youget bored, you canalways use the "enzyme" for its original purpose!
Volume 68
Number 4
April 1991
287
