SCIENTIFIC AMERICAN ENZYME MOLECULE /966 The Three-dimensional Structure of an Enzyme Molecule The arrangement of atoms in an enzyme molecule has been worked out for the first time. The enzyme is lysozyme, which breaks open cells of bacteria. The study has also shown how lysozyme performs its task by David C. Phillips ne day in 1922 Alexander Flem- determined and whose properties are corporated into a polypeptide chain a ing was suffering from a cold. understood in atomic detail. Among residue, and each residue has its own O This is not unusual in London, these properties is the way in which the characteristic side chain. The 129-resi- but Fleming was a most unusual man enzyme combines with the substance on due lysozyme molecule is cross-linked and he took advantage of the cold in a which it acts—a complex sugar in the in four places by disulfide bridges characteristic way. He allowed a few wall of the bacterial cell. formed by the combination of sulfur- drops of his nasal mucus to fall on a Like all enzymes, lysozyme is a pro- containing side chains in different parts culture of bacteria he was working with tein. Its chemical makeup has been of the molecule [see illustration on op- and then put the plate to one side to established by Pierre Jolles and his posite page}. see what would happen. Imagine his colleagues at the University of Paris The properties of the molecule cannot excitement when he discovered some and by Robert E. Canfield of the Co- be understood from its chemical con- time later that the bacteria near the lumbia University College of Physicians stitution alone; they depend most criti- mucus had dissolved away. For a while and Surgeons. They have found that cally on what parts of the molecule are he thought his ambition of finding a each molecule of lysozyme obtained brought close together in the folded universal antibiotic had been realized. from egg white consists of a single three-dimensional structure. Some form In a burst of activity he quickly estab- polypeptide chain of 129 amino acid of microscope is needed to examine the lished that the antibacterial action of subunits of 20 different kinds. A pep- structure of the molecule. Fortunate- the mucus was due to the presence in tide bond is formed when two amino ly one is effectively provided by the it of an enzyme; he called this substance acids are joined following the removal of techniques of X-ray crystal-structure lysozyme because of its capacity to lyse, a molecule of water. It is customary to analysis pioneered by Sir Lawrence or dissolve, the bacterial cells. Lyso- call the portion of the amino acid in- Bragg and his father Sir William Bragg. zyme was soon discovered in many tis- sues and secretions of the human body, in plants and most plentifully of all in ALA ALANINE GLY GLYCINE PRO PROLINE the white of egg. Unfortunately Flem- ARG ARGININE HIS HISTIDINE SER SERINE ing found that it is not effective against ASN ASPARAGINE ILEU ISOLEUCINE THR THREONINE the most harmful bacteria. He had to ASP ASPARTIC ACID LEU LEUCINE TRY TRYPTOPHAN wait seven years before a strangely CYS CYSTEINE LYS LYSINE TYR TYROSINE similar experiment revealed the exis- GLU GLUTAMIC ACID MET METHIONINE VAL VALINE tence of a genuinely effective antibi- GLN GLUTAMINE PHE PHENYLA1 ANINE otic: penicillin. TWO-DIMENSIONAL MODEL of the lysozyme molecule is shown on the opposite page. Nevertheless, Fleming's lysozyme has Lysozyme is a protein containing 129 amino acid subunits, commonly called residues (see proved a more valuable discovery than key to abbreviations above). These residues form a polypeptide chain that is cross-linked at he can have expected when its prop- four places by disulfide (-S-S-) bonds. The amino acid sequence of lysozyme was deter- erties were first established. With it, mined independently by Pierre Jolles and his co-workers at the University of Paris and by for example, bacterial anatomists have Robert E. Canfield of the Columbia University College of Physicians and Surgeons. The been able to study many details of bac- three-dimensional structure of the lysozyme molecule has now been established with the help of X-ray crystallography by the author and his colleagues at the Royal Institution in terial structure [see "Fleming's Lyso- London. A painting of the molecule's three-dimensional structure appears on pages 80 and zyme," by Robert F. Acker and S. E. 81. The function of lysozyme is to split a particular long-chain molecule, a complex sugar, Hartsell; SCIENTIFIC AMERICAN, June, found in the outer membrane of many living cells. Molecules that are acted on by enzymes I960]. It has now turned out that are known as substrates. The substrate of lysozyme fits into a cleft, or pocket, formed by the lysozyme is the first enzyme whose three-dimensional structure of the lysozyme molecule. In the two-dimensional model on three-dimensional structure has been the opposite page the amino acid residues that line the pocket are shown in dark green. 78 30 >. JL MAIN CHAIN NITROGEN CARBON OXYGEN SULFUR SIDE CHAIN CARBON HYDROGEN BOND THREE-DIMENSIONAL MODEL of the ly- sozyme molecule, painted by Irving Geis, is based on an actual model assembled at the Royal Institution by the author and his col- leagues. The painting enables one to trace and distinguish between the chemical bonds that hold together the main polypeptide chain and the bonds in the 129 side chains, one for each amino acid residue. The mole- cule is folded so as to form a cleft that holds the substrate molecule while it is being broken in two. The painting on the next page shows how the substrate fits into the cleft. The red balls represent oxygen atoms that are important in splitting the substrate. \ \ L The difficulties of examining mole- according to whether the waves are in 1015 identical molecules in a regular cules in atomic detail arise, of course, or out of phase—in or out of step— array; in effect the molecules in such a from the fact that molecules are very with one another. (This effect is seen crystal diffract the X radiation as though small. Within a molecule each atom most easily in light waves scattered they were a single giant molecule. The is usually separated from its neighbor by a regularly repeating structure, such crystal acts as a three-dimensional dif- by about 1.5 angstrom units (1.5 X 10'8 as a diffraction grating made of lines fraction grating, so that the waves scat- centimeter). The lysozyme molecule, scribed at regular intervals on a glass tered by them are confined to a number which contains some 1,950 atoms, is plate.) In the second stage of image of discrete directions. In order to obtain about 40 angstroms in its largest di- formation, according to Abbe, the ob- a three-dimensional image of the struc- mension. The first problem is to find a jective lens of the microscope collects ture the intensity of the X rays scattered microscope in which the atoms can be the diffracted waves and recombines in these different directions must be resolved from one another, or seen sep- them to form an image of the object. measured, the phase problem must be arately. Most important, the nature of the im- solved somehow and the measurements The resolving power of a microscope age depends critically on how much of must be combined by a computer. depends fundamentally on the wave- the diffraction pattern is used in its The recent successes of this method length of the radiation it employs. In formation. in the study of protein structures have general no two objects can be seen sep- depended a great deal on the develop- arately if they are closer together than X-Ray Structure Analysis ment of electronic computers capable about half this wavelength. The short- of performing the calculations. They est wavelength transmitted by optical In essence X-ray structure analysis are due most of all, however, to the microscopes (those working in the ul- makes use of a microscope in which discovery in 1953, by M. F. Perutz of traviolet end of the spectrum) is about the two stages of image formation have the Medical Research Council Labora- 2,000 times longer than the distance been separated. Since the X rays can- tory of Molecular Biology in Cambridge, between atoms. In order to "see" atoms not be focused to form an image di- that the method of "isomorphous re- one must use radiation with a much rectly, the diffraction pattern is re- placement" can be used to solve the shorter wavelength: X rays, which have corded and the image is obtained from phase problem in the study of protein a wavelength closely comparable to it by calculation. Historically the meth- crystals. The method depends on the interatomic distances. The employment od was not developed on the basis of preparation and study of a series of pro- of X rays, however, creates other dif- this reasoning, but this way of regard- tein crystals into which additional heavy ficulties: no satisfactory way has yet ing it (which was first suggested by atoms, such as atoms of uranium, have been found to make lenses or mirrors Lawrence Bragg) brings out its essen- been introduced without otherwise af- that will focus them into an image. The tial features and also introduces the fecting the crystal structure. The first problem, then, is the apparently im- main difficulty of applying it. In re- successes of this method were in the possible one of designing an X-ray cording the intensities of the diffracted study of sperm-whale myoglobin by microscope without lenses or mirrors.