UNTIL RECENTLY only a full-time scientific worker would have been likely to undertake to build a model of an enzyme, partly because these giant molecules that catalyze the chemical reactions of the living cell are highly complex and partly because the task is quite expensive with most model-building kits. Frank H. Clarke (14 Long Pond Road, Armonk, N.Y. 10504) decided to see if the project could be brought within the reach of amateurs in terms of both skill and cost. He found that it could. Clarke, a medicinal research chemist, is deputy director of the chemistry division of Ciba-Geigy Corporation. He recently received a patent for the novel features of the model-building components he describes as follows.

"There are two major kinds of model for illustrating molecular architecture. The skeletal model indicates the centers of the atoms and the bonds that join them; it resembles the kind of line drawing an organic chemist would make to define a molecular structure. The space-filling model shows both the shape of the molecule and the volume it occupies. It is particularly useful for suggesting how one molecule interacts with another.

"Up to now it has not been very practical even for the professional scientist to build a model that is both skeletal and space-filling. In the method I have devised it is possible to make such a model, and with inexpensive components. The finished model not only is instructive as a guide for understanding the details of modern biochemistry but also is a handsome object to have in the home.

"The method can be applied to any biological molecule for which the atomic architecture is fully known. The enzyme lysozyme is a good example. A model I have made portrays the catalytically active site of alpha chymotrypsin a proteolytic (protein-splitting) enzyme [see illustration at left]. The first step in learning to make such a model is to consider the structure of peptides and the bonds that give rise to it.

"A peptide bond is constructed by the linking of two amino acid molecules through the carboxyl (COOH) group of one and the amino (NH) group of another. A polypeptide is a long-chain polymer made in this way from the 20 or so natural amino acids. The chain is arranged so that the carboxyl group of the -first amino acid forms part of an amide (peptide) bond with the amino group of the second and so on. The final amino acid in the chain has a free carboxyl group. It is convenient to build a model in the same way that nature builds a protein. Each chain begins with a free amino group of the first amino acid and ends with the free carboxyl group of the last amino acid.

"As the polypeptide chain grows, the successive amino acids become fixed in a particular configuration that can be described in two ways. One is to give the spatial coordinates of each atom. The other, which is possible because the distances between successive atoms are known, is to work in terms of the dihedral angles around the bonds that join the atoms in the chain.

"For the molecular hobbyist it is convenient to use components cut to predetermined lengths and to fix the angles around the successive bonds. Nearly all peptide bonds are trans and planar, n meaning that hydrogen and carbonyl oxygen (CO) are on opposite sides of the bond. Hence it is necessary only to fix -the two dihedral angles around each alpha carbon atom and then to link each amino acid successively with its predecessor in the chain to form trans and planar amide bonds.

"As the chain is extended small errors are often multiplied. It is therefore necessary to have additional means of determining the shape of the chain. Skeletal drawings based on the horizontal coordinates of the chain segments are helpful. Moreover, since all proteins have a secondary structure determined partly by hydrogen bonds, these bonds can also help the model builder to work out the conformation of the molecule. When the main chain has been made, it is mounted on a base and the side chains are added. Finally, space fillers are attached to round out the molecule.

"I make models with components I obtain commercially and then modify. Two of the basic components are made by Prentice-Hall, Inc. (Englewood Cliffs, N.J. 07G32), namely Framework Molecular Model tubes and straight aluminum connecting pins. Two others are available from Science Related Materials, Inc. (P.O. Box 1422, Janesville, Wis. 53545), namely color-coded Minit atom centers and expanded-polystyrene balls.

"The modified components I make are unique in two ways. First, they are color-coded, so that not only the plastic atom centers but also the connecting tubes are colored to identify the atoms that are joined. Second, the tubes have tapered cavities so that they can be pressed onto the arms of the atom centers to fix the dihedral angles. Each atom and bond of the completed model is readily identified by its color: blue for nitrogen, red for oxygen, black for carbon, yellow for sulfur and white for hydrogen. The polystyrene space fillers are also color-coded to represent the individual atoms.

"I work at a scale of 12.5 millimeters per angstrom. It is the same as the scale of the well-known Corey-Pauling-Koltun (CPK) space-filling models. The two sets of models can therefore be used interchangeably.

"For the model builder the most important consideration is the overall form of the molecule, particularly when the molecule is a complex bio-organic compound. It is not necessary to locate the center of each hydrogen atom. Moreover, the distance between carbon-hydrogen bonds can be made longer than the scale distance between the atom centers. The distance between carbon-carbon bonds, however, must be exact because the length and shape of the chain are important.

"Even so, chains are frequently chains are normally bent back on themselves. The angles of the bends are important, but often the individual distances between atoms need not be exact to build a good representation of a complete peptide. Thus the connecting tubes of the color-coded skeletal models have only three different lengths (12.5, 14.5 and 21.7 millimeters). Those lengths suffice for a wide variety of models of bio-organic compounds. When the tubes are connected to the Minit atom centers, they provide interatomic distances representing 1.36 angstroms (for short bonds, such as those in aromatic rings), 1.54 angstroms (for bonds of medium length, such as those in aliphatic carbon compounds) and 1.81 angstroms (for longer bonds, such as those involving sulfur atoms). The color combinations of the various lengths are indicated in the accompanying illustration [at right].

]. Yellow Testors Pla enamel paint is used to color black and white tubes for S-C and S-H bonds.

"The color-coded tubes with tapered cavities are not yet available commercially. It is easy to make them, however, by cutting to appropriate lengths the Framework Molecular Model tubes. Hold a tube in a suitable hand vise and drill the tapered cavities with a hand power drill fitted with an eighth-inch bit that has been ground to a taper with an emery stone. Alternatively a tapered cavity can be made by pushing the tube onto the hot No. 5 conical tip of a Weller soldering iron.

"The color-coded Minit atom centers are most convenient for use with these tubes. The atom centers are inexpensive and are supplied in a variety of colors with tetrahedral and planar-trigonal arrangements of the valence arms. Special trigonal carbon and nitrogen centers are supplied for building the amide bonds of peptides.

"In addition the short, straight aluminum connecting rods supplied by Prentice-Hall are convenient for forming the many different angles involved in the hydrogen bonds of polypeptides. They are easily bent to the proper angle with two short lengths of brass tubing, such as those that hold the ink in certain ballpoint pens. A sturdy base and stiff wire rods are needed to support a complex model. The rods can be made from wire coat hangers.

"The space-filling polystyrene balls come in two diameters, one inch (25.4 millimeters) for hydrogen atoms and 1.37 inches (34.8 millimeters) for oxygen and sulfur atoms. The hydrogen atom is exactly to scale. The oxygen atom is somewhat larger than the scale calls for and the sulfur atom is a little smaller, but the discrepancies are not large.

"The bottom third of the hydrogen atom is painted the color of the atom to which it is joined: black for carbon, blue for nitrogen and red for oxygen. Carbonyl-oxygen atoms are painted red with a black base. The sulfur atoms are yellow, with black for the carbon portion of a carbon-sulfur bond. Since the sulfur atoms are divalent, the spheres must be shaped. One must also make holes in the base of each hydrogen and carbonyloxygen atom so that it will fit neatly over the skeletal tube.

"In sum, the components of a modelbuilding kit that is color-coded and both skeletal and space-filling are simple and either available or easily prepared. They are so inexpensive and the assembled model is so attractive that it is preferable to make a new model from fresh components rather than to disassemble an existing model. Entire collections of molecules can be easily assembled and employed in comparative studies of molecular architecture.

"The points that must be taken into account in assembling a model of a polypeptide are the construction of the amide bonds, the arrangement of atoms around the asymmetric alpha carbon atom, the measurement of dihedral angles and the construction of the hydrogen bonds. As I have mentioned, each amino acid of the peptide chain is joined to its neighbor by an amide bond. The N-H is trans and planar to the carbonyl oxygen. The amide bond is formed between two Minit atom centers with two double bars [see illustration at left]. The angle opposite the pair of double bars is 114 degrees. Short connecting tubes serve for the N-C bond and for the carbonyl group. The other tubes are of medium length.

"To make a peptide chain one first prepares the amino acid units and then joins them in sequence with the amide bonds. An amino acid unit is depicted in the accompanying illustration [at right]. It has three components: the N-H, or amino, component ["c" in illustration], the alpha carbon atom ["d"] and the carbonyl component ["e"]. The alpha carbon atom is shown attached to its alpha hydrogen atom. It is advisable to put on the tube for the hydrogen atom a label with a number to indicate the particular amino acid that is to be represented. Avery self-adhesive labels are convenient for this purpose, provided that you cover each one with a small piece of transparent tape to keep it from unwrapping. The number is particularly important because in model building the distinguishing side chain R is left off initially, so that only the vacant arm indicates the stereochemistry.

"To assemble the amino and carbonyl components insert the appropriate tubes in vertical holes you have drilled in a board so that one end of each tube projects above the board. Then coat the tip of the appropriate arm of a Minit center with '5 Minute' epoxy cement (freshly mixed) and insert it in a tube. Repeat the process until all the components have been assembled.

"Finally, two of the arms of the alpha carbon are coated with epoxy cement and inserted in the black tubes of the amino component and the carbonyl component respectively. Take care to maintain the alpha carbon in the orientation shown. The completed unit ["b" in illustration] has the N-H on the left and the CO on the right; the hydrogen of the alpha carbon points down and the vacant arm points up. The unit now has the correct absolute stereochemistry.

"The next step is to determine and fix the dihedral angles around the alpha carbon atom. The angles are called phi and psi [see illustration at left]. Phi is the angle between successive carbonyl carbon atoms around the nitrogen-alpha carbon (N-CA) bond. Psi is the angle between successive nitrogen atoms around the CO-CA bond.

"To measure phi one sights along the N-CA bond with the carbonyl carbon pointed to the right. Phi is the angle the vacant arm on the nitrogen (blue) atom center makes with the CO-CA bond. Since the vacant arm is so short, more accuracy can be obtained by putting a thin drinking straw over the arm to lengthen it. The psi angle is measured by sighting along the CO-CA bond with the nitrogen having the vacant arm pointing to the right. Psi is the angle the CO-N bond makes with the CA-N bond.

"Dihedral angles of proteins are recorded in various ways. It is important to check which angle is measured around the N-CA bond and which one is measured around the CO-CA bond, whether an angle of zero degrees is cis or trans and whether a positive angle is measured clockwise or counterclockwise.

"When you have measured the dihedral angles, press the tubes firmly onto the arms to fix the atoms in position. The angles are easily adjusted while the cement is soft, but after it has set each bond soon becomes permanent.

"Approximately 10 amino acid units are first joined to form segments of a polypeptide chain, and later the segments are joined. Make the amide bond by coating the vacant arm of amino acid unit No. 2 with epoxy cement and inserting the arm into the nitrogen-bond cavity of the C-N tube attached to the CO carbon center of amino acid No. 1. Do it so that the amide bond is trans and planar The amine of unit 3 is then attached to the carbonyl of unit 2 and so on until the segment is complete.

"Often it develops that when the chain folds back on itself, hydrogen bonds are formed between amido hydrogens and carbonyl oxygens of the same chain. Hydrogen bonds are also formed between adjacent chains, and they provide the secondary structure that keeps the polypeptide in a particular shape. For the model builder hydrogen bonds provide structural strength, so that fewer outside supports have to be supplied.

"In a polypeptide molecule many of the hydrogen bonds are between an amido hydrogen and the carbonyl of neighboring amide. Often the line joining the carbonyl, the hydrogen atom and the nitrogen atom follows a zigzag course. When the hydrogen bond is linear, the distance between the center of the nitrogen atom and the center of the oxygen atom is three angstroms. On the scale of 12.5 millimeters per angstrom the distance is represented in the model by a distance of 37.5 millimeters [see illustration at right].

"To make such an arrangement the van der Waals radius of oxygen is represented by a long tube, the CO double bond by a short tube and the NH by a tube of medium length. The long tube is joined to the CO and the NH by Framework Molecular Model linear aluminum pins In the assembled linear hydrogen bond the distance between the center of the carbon of the carbonyl and the center of the nitrogen of the amide group is 54 millimeters. It is a relatively simple matter to extend this construction technique to form hydrogen bonds that are not linear. For this purpose you bend the aluminum connectors to the proper angles before joining them to the plastic tubes.

"Occasionally a single carbonyl oxygen is involved in two hydrogen bonds. Make them with a planar trigonal oxygen [see illustration at left]. In this instance the van der Waals radius of oxygen is represented by a tube of medium length. The aluminum connectors can be either straight or bent as depicted in the upper and lower parts of the illustration respectively. The same principles are easily adapted to form hydrogen bonds between hydroxyl groups or between a hydroxyl group and the nitrogen atom of an imidazole group of histidine. For the latter a short blue tube represents the van der Waals radius of nitrogen.

"The arms of the aluminum connectors are smaller in diameter than the arms of the Minit atom centers. The pins fit the Framework Molecular Model tubes without a tapered cavity. Accordingly the white ends of the tubes representing OH and NH are not drilled, and neither are the ends of the long red tubes. One end of each medium-length red tube is drilled to fit the arm of a Minit center. Carbonyl tubes for amide bonds are drilled only at the black end, but CO tubes for hydroxyl groups are drilled at both ends.

"When the main chain, with its hydrogen bonds, is assembled, the side chains are constructed and added. For this task it is most convenient to prepare plans of the horizontal coordinates of the nonhydrogen atoms of small segments of the main chain. The plans become quite crowded if the segments are longer than about 10 amino acid units.

"After the skeletal model is complete it can be partly or completely converted to a space-filling model by adding the painted polystyrene balls. The illustration [right] shows the space-filling hydrogen atom with its painted bottom third.

"The use of models of enzymes enables the medicinal chemist tostudy the interaction of small molecules with complex ones, to design enzyme inhibitors and to gain understanding of the intricate mechanisms of complex bio-organic processes. Eventually studies such as these will aid directly in the discovery of new medicinal agents."

Bibliography

THE THREE-DIMENSIONAL STRUCTURE OF AN ENZYME MOLECULE. David C. Phillips in Scientific American, Vol. 215, No. 5, pages 78-90; November, 1966.

STRUCTURE OF CRYSTALLINE ALPHA-CHYMOTRYPSIN: V. THE ATOMIC STRUCTURE OF TOSYL-ALPHA-CHYMOTRYPSIN AT 2 A RESOLUTION, J. J. Birktoft and D. M. Blow in Journal of Molecular Biology, Vol.68, No. 2, pages 187-240; July 21, 1972.

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