John F. Sebastlan and John C. Butkus Miami University Oxford. Ohio 45056
A Space-Filling Model of the Active Site Region of Carboxypeptidase A
Table 1. Amino Acid Residues Used in Constructing One of the most important developments in the Active Site Region study of enzyme catalysis has been the elucidation of three-dimensional stru&ures of crystalline enzymes hg 266 Ser 65 Asp 123 hsn 192 Phe 238 Tyr 248 Tyr 66 Leu 121 Arg 193 re,, 239 Lyr 267 Pile 249 Gln X-ray crystallography. Although there may he significant 194 Ser 240 Tyr 268 Tin 125 Leu 67 Gly 250 A h differences between solution i d crystal structures, much 269 Phe 68 Ile 126 Trp 195 Ile 241 GIT 251 Ser useful information has been provided by the latter. 69 His 127 \rg 196 Xis 242 Ser 270 Glu 252 Gly 243 Ile 271 Leu 70 Ser I28 LYS 197 Ser 253 Gly Carhoxypeptidase A (CPA) is a zinc-containing proteo71 Arg 129 T h r 19s T Y ~ 211 Ile 272 i r g 254 Ser lytic enzyme, which catalyzes the hydrolysis of carhoxy-ter199 Ser 245 Thr 273 i s p 72 Gln 255 Ile 73 Trp 142 Asp 200 Gln 246 Thr 274 Thr 256 Asp minal peptide bonds in proteins and peptides. The en74 11. 143 Ala 201 Leu 247 Ile 275 Gly 257 Trp zyme exhibits esterase activity as well. CPA is involved in 276 hrz 75 Thr 144 ,\an 202 Leu 2 % Ser protein digestion in the duodenum and occurs in the pan145 Arg 203 Leu 277 Tyr 146 Asn 204 Tyr 278 Gly creatic iuice in the form of a zvmoeen. ~ r o c a r b o x v ~ e ~ t i d 147 Trp 205 Pro 279 Phe ase A. ~ a r h o x ~ p e p t i d a sAe is the first metalloenGm> for 148 A ~ D 206 Tyr 280 Leu 207 Gly 281 Leu which the X-ray structure (at 2.0 A resolution) and amino 208 Tyr acid sequence have been determined. Several excellent re209 Thr views on CPA have appeared recently (1-5). In connection with some studies on the inhibition of CPA, we found it desirable to build a three-dimensional Corey-Pauling-Koltun (CPK) space-filling model of the active site region of the enzyme. We have suhsequently used this model for demonstration purposes in our hioPlexiglas sheets were centered over each other on a drillchemistry and organic chemistry classes with considerable ing mill and the graph-paper template was taped onto the success. Published (4, 7) three-dimensional representaPlexiglas sheet. Holes of The-in. diameter were drilled tions of CPA were consulted in determining which amino through the points corresponding to selected backbone acid residues should he used in constructing the active carbonyl oxygen atoms (see below) indicated on the temsite region (see the table). The orthogonal atomic coordiplate. nates for CPA have been tabulated (5) and these were To achieve maximum support of the model with minisuhsequently scaled to 1.25 cm/A. mum interference from supporting wires, holes were A rectangular support frame, measuring 30 X 30 X 35314 drilled throueh the backbone carhonvl oxveen atoms of in., was constructed from square aluminum tubing (1% X the following-residues: 66, 72, 75, 123: 129,.ih2, 145, 148, 1% in.). The molecular model was supported by two %-in. 192. 196. 197. 200. 201. 203. 205. 207. 209. 239.. 240.. 243.. Plexiglas sheets; one was mounted on the top of the frame 250; 253; 255; 257; 266, 271, 276, and 281. Oxygen atom and the other on the hase. The sheets were attached with 243 was connected only to the top Plexiglas sheet. The screws and glued to triangular plywood comers (3I4-in. carhonyl oxygens were chosen as the atoms to pass the thick) which were, in turn, screwed to the frame. Prior to wires through because their slotted design makes them mounting the Plexiglas sheets to the frame and plywood, a particularly suitable for looping and securing the wire. template was constructed by plotting on graph paper the The support wire was looped twice to prevent the model x-z plane of the active site region. This plot included all from gradually sliding down. The carhonyl oxygen atoms atoms (except hydrogen) used in building the model. Both were positioned in the y direction hy use of a meter stick cut so that it could he placed snugly between the Plexiglas sheets supporting the model. The supporting wires were attached to the Plexiglas sheets by fasteners constructed from %-in. square aluminum tuhing cut into %-in. segments. A %-in. hole was drilled through the center of the fastener and through the opposite side. The fasteners were then attached to the inside of the Plexiglas sheet with 4-40 X %-in. round head machine screws with hex nuts (Fig. 1). Side chains were attached and oriented, after hanging the polypeptide backbone. A similar procedure was used by Yankeelov and Coggins (8) in their construction of a CPK model of myoglohin. Aluminum H channels were placed above the top Plexiglas sheet and below the hase sheet to prevent sagging and to give added support to the frame. A tetrahedral zinc ion was constructed from aluminum and iron sheets and pop rivets. A van der Waals radius of 1.30 A, converted to the CPK scale, was chosen as the distance from the center of the tetrahedron to the CPK sockets attached to the comers. See Figure 2 for details of construction. Once the model zinc ion was placed into the active site and attached to the appropriate ligands (His 69, His 196, and Gln 72), it was found that His 69 deviatFigure 1. Attachment of fasteners to Plexiglas sheet. Wires supporting model are tied to thefasteners. ed slightly from the published coordinates. This most ~
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660 / Journal of Chemical Education
Figure 4. Pocket of active site.
probably results from using a regular tetrahedron for zinc rather than a distorted tetrahedron as is suggested by the X-ray data. After building the model, additional supporting structures were constructed from %-in. diameter steel rods with an approximate length of 20 in., test tuhe clamps, and thumb tacks. The test tuhe clamps were attached to one end of the rod and the thumh tacks then soldered onto the clamps (see Fig. 2). The thumh tacks were attached to selected hydrogen atoms and the rods clamped to the frame of the model as shown in Figure 3. The "dead-end" pocket of the CPA active site is easily seen in Figure 4. One can clearly demonstrate why carhoxypeptidase is an exopeptidase by simply placing a substrate model in the active site with the R group of the C-terminal residue placed in the pocket. In Figure 5 benzoyl glycyl-L-phenylalanine (BGP) has been placed a t the active site with the phenyl group of the phenylalanine residue located in the pocket and carhoxylate moiety in close proximity to Arg 145. This placement of the suhstrate is consistent with X-ray data (5). In addition, one can use this model to demonstrate the various conformational changes which occur upon binding of a substrate to CPA. For example, Tyr 248 is known (from X-ray data) to move about 12 A when a substrate is bound to CPA. This movement can he seen by comparing Figures 4 and 5.
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I.
A Space-Filling Model of the Active Site Region of Carboxypeptidase A
Table 1. Amino Acid Residues Used in Constructing One of the most important developments in the Active Site Region study of enzyme catalysis has been the elucidation of three-dimensional stru&ures of crystalline enzymes hg 266 Ser 65 Asp 123 hsn 192 Phe 238 Tyr 248 Tyr 66 Leu 121 Arg 193 re,, 239 Lyr 267 Pile 249 Gln X-ray crystallography. Although there may he significant 194 Ser 240 Tyr 268 Tin 125 Leu 67 Gly 250 A h differences between solution i d crystal structures, much 269 Phe 68 Ile 126 Trp 195 Ile 241 GIT 251 Ser useful information has been provided by the latter. 69 His 127 \rg 196 Xis 242 Ser 270 Glu 252 Gly 243 Ile 271 Leu 70 Ser I28 LYS 197 Ser 253 Gly Carhoxypeptidase A (CPA) is a zinc-containing proteo71 Arg 129 T h r 19s T Y ~ 211 Ile 272 i r g 254 Ser lytic enzyme, which catalyzes the hydrolysis of carhoxy-ter199 Ser 245 Thr 273 i s p 72 Gln 255 Ile 73 Trp 142 Asp 200 Gln 246 Thr 274 Thr 256 Asp minal peptide bonds in proteins and peptides. The en74 11. 143 Ala 201 Leu 247 Ile 275 Gly 257 Trp zyme exhibits esterase activity as well. CPA is involved in 276 hrz 75 Thr 144 ,\an 202 Leu 2 % Ser protein digestion in the duodenum and occurs in the pan145 Arg 203 Leu 277 Tyr 146 Asn 204 Tyr 278 Gly creatic iuice in the form of a zvmoeen. ~ r o c a r b o x v ~ e ~ t i d 147 Trp 205 Pro 279 Phe ase A. ~ a r h o x ~ p e p t i d a sAe is the first metalloenGm> for 148 A ~ D 206 Tyr 280 Leu 207 Gly 281 Leu which the X-ray structure (at 2.0 A resolution) and amino 208 Tyr acid sequence have been determined. Several excellent re209 Thr views on CPA have appeared recently (1-5). In connection with some studies on the inhibition of CPA, we found it desirable to build a three-dimensional Corey-Pauling-Koltun (CPK) space-filling model of the active site region of the enzyme. We have suhsequently used this model for demonstration purposes in our hioPlexiglas sheets were centered over each other on a drillchemistry and organic chemistry classes with considerable ing mill and the graph-paper template was taped onto the success. Published (4, 7) three-dimensional representaPlexiglas sheet. Holes of The-in. diameter were drilled tions of CPA were consulted in determining which amino through the points corresponding to selected backbone acid residues should he used in constructing the active carbonyl oxygen atoms (see below) indicated on the temsite region (see the table). The orthogonal atomic coordiplate. nates for CPA have been tabulated (5) and these were To achieve maximum support of the model with minisuhsequently scaled to 1.25 cm/A. mum interference from supporting wires, holes were A rectangular support frame, measuring 30 X 30 X 35314 drilled throueh the backbone carhonvl oxveen atoms of in., was constructed from square aluminum tubing (1% X the following-residues: 66, 72, 75, 123: 129,.ih2, 145, 148, 1% in.). The molecular model was supported by two %-in. 192. 196. 197. 200. 201. 203. 205. 207. 209. 239.. 240.. 243.. Plexiglas sheets; one was mounted on the top of the frame 250; 253; 255; 257; 266, 271, 276, and 281. Oxygen atom and the other on the hase. The sheets were attached with 243 was connected only to the top Plexiglas sheet. The screws and glued to triangular plywood comers (3I4-in. carhonyl oxygens were chosen as the atoms to pass the thick) which were, in turn, screwed to the frame. Prior to wires through because their slotted design makes them mounting the Plexiglas sheets to the frame and plywood, a particularly suitable for looping and securing the wire. template was constructed by plotting on graph paper the The support wire was looped twice to prevent the model x-z plane of the active site region. This plot included all from gradually sliding down. The carhonyl oxygen atoms atoms (except hydrogen) used in building the model. Both were positioned in the y direction hy use of a meter stick cut so that it could he placed snugly between the Plexiglas sheets supporting the model. The supporting wires were attached to the Plexiglas sheets by fasteners constructed from %-in. square aluminum tuhing cut into %-in. segments. A %-in. hole was drilled through the center of the fastener and through the opposite side. The fasteners were then attached to the inside of the Plexiglas sheet with 4-40 X %-in. round head machine screws with hex nuts (Fig. 1). Side chains were attached and oriented, after hanging the polypeptide backbone. A similar procedure was used by Yankeelov and Coggins (8) in their construction of a CPK model of myoglohin. Aluminum H channels were placed above the top Plexiglas sheet and below the hase sheet to prevent sagging and to give added support to the frame. A tetrahedral zinc ion was constructed from aluminum and iron sheets and pop rivets. A van der Waals radius of 1.30 A, converted to the CPK scale, was chosen as the distance from the center of the tetrahedron to the CPK sockets attached to the comers. See Figure 2 for details of construction. Once the model zinc ion was placed into the active site and attached to the appropriate ligands (His 69, His 196, and Gln 72), it was found that His 69 deviatFigure 1. Attachment of fasteners to Plexiglas sheet. Wires supporting model are tied to thefasteners. ed slightly from the published coordinates. This most ~
~~
660 / Journal of Chemical Education
Figure 4. Pocket of active site.
probably results from using a regular tetrahedron for zinc rather than a distorted tetrahedron as is suggested by the X-ray data. After building the model, additional supporting structures were constructed from %-in. diameter steel rods with an approximate length of 20 in., test tuhe clamps, and thumb tacks. The test tuhe clamps were attached to one end of the rod and the thumh tacks then soldered onto the clamps (see Fig. 2). The thumh tacks were attached to selected hydrogen atoms and the rods clamped to the frame of the model as shown in Figure 3. The "dead-end" pocket of the CPA active site is easily seen in Figure 4. One can clearly demonstrate why carhoxypeptidase is an exopeptidase by simply placing a substrate model in the active site with the R group of the C-terminal residue placed in the pocket. In Figure 5 benzoyl glycyl-L-phenylalanine (BGP) has been placed a t the active site with the phenyl group of the phenylalanine residue located in the pocket and carhoxylate moiety in close proximity to Arg 145. This placement of the suhstrate is consistent with X-ray data (5). In addition, one can use this model to demonstrate the various conformational changes which occur upon binding of a substrate to CPA. For example, Tyr 248 is known (from X-ray data) to move about 12 A when a substrate is bound to CPA. This movement can he seen by comparing Figures 4 and 5.
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I.
