Section 26.1.4.3. Low-resolution binding studies of lysozyme with GlcNAc and other sugars

In the first binding studies of lysozyme with GlcNAc, crystals were soaked overnight in solutions of 0.15 M GlcNAc in the standard crystallization medium, and a 15° precession photograph was recorded by LNJ. The changes in intensities of reflections compared to a native lysozyme photograph were extremely small, much smaller than those observed with the heavy-atom derivatives, but were sufficiently promising to encourage us to collect three-dimensional data to 6 Å resolution. Data were collected on the linear diffractometer using the three counters in the flat-cone setting over a period of about 20 h, as described for the heavy-atom derivatives. The data were processed using the programs of ACTN and VRS on the Elliott 803B computer. The 6 Å difference-Fourier map, using the phases from the improved set of heavy-atom derivatives, was obtained at the end of October 1964. It showed a single rather elongated peak which, when superimposed onto the 6 Å model, was located in the cleft in the enzyme surface between the two domains (Johnson & Phillips, 1965).

The power of the difference-Fourier technique in protein crystallography was immediately demonstrated by this first 6 Å electron-density difference map for the lysozyme–inhibitor complex, as had also been demonstrated earlier in the work (Stryer et al., 1964) on the binding of azide to sperm-whale myoglobin. Once a protein structure had been solved, it was apparent that ligand-binding sites could be established with ease. Following the GlcNAc result, 6 Å binding studies were repeated with a number of other compounds (Blake et al., 1967). Kinetic studies using the turbidometric assay were carried out with each of the compounds in order to establish the mode of inhibition of lysozyme activity. We were fortunate in being able to use the skills of JWHO, a carbohydrate chemist who had been at the Royal Institution for many years. JWHO had synthesized 6-iodo-6-deoxy-N-acetyl β-methylglucosaminide, a compound which was found to inhibit more powerfully than GlcNAc itself. The low-resolution binding study showed a stronger and more compact peak at the catalytic site than that observed with GlcNAc, but it was not possible to resolve the iodine and hence identify the six positions of the sugar.

By January 1965, we had been given a sample of the disaccharide di-N-acetylchitobiose, (GlcNAc)₂, sent by John Rupley. Efficiency of data collection and map production had increased. Data collection was started on 19 January and the map was obtained by 28 January. Other results followed with N-acetylmuramic acid (a gift from R. W. Jeanloz) and the disaccharide N-acetylglucosamine β-(1,4)-N-acetylmuramic acid (GlcNAc-MurNAc – a gift from N. Sharon) (Fig. 26.1.4.2).

Figure 26.1.4.2| top | pdf | Inhibitor molecules of lysozyme (Blake et al., 1967). (a) N-acetylglucosamine; (b) N-acetylmuramic acid; (c) 6-iodo-α-methyl-N-acetylglucosaminide; (d) α-benzyl-N-acetylmuramic acid; (e) di-N-acetylchitobiose; (f) N-acetylglucosaminyl-N-acetylmuramic acid; (g) tri-N-acetylchitotriose.

As part of these studies, penicillin V and p-iodophenoxymethyl penicillin potassium salt were also investigated. They were observed to inhibit lysozyme. Crystallographic studies showed that the penicillins did indeed bind to lysozyme in the catalytic cleft, but at a site remote from the GlcNAc binding site (Johnson, 1967). As established shortly after this result was obtained in 1965, penicillin exerts its potent antibiotic activity by inhibition of the enzymes responsible for the biosynthesis of the peptide cross-linking component of the bacterial cell wall (Tipper & Strominger, 1965; Wise & Park, 1965). An original suggestion that penicillin might resemble MurNAc turned out to be incorrect (Collins & Richmond, 1962). The interactions of penicillin with lysozyme are probably fortuitous, but were not fully investigated.

By the end of May 1965, we were ready to move on to 2 Å data collection, a formidable task that required fourteen crystals and more than two weeks continuous data collection on the multiple-counter linear diffractometer. Data for the lysozyme–GlcNAc complex were completed first, and the map was available around October 1965. The electron density was puzzling, and an interpretation was not possible until the high-resolution results with the trisaccharide were available.