Structural and spectroscopic studies of lysozyme

The characterisation of productive enzyme-substrate complexes stabilised by sub-zero enzymologic methods in the crystalline state is considered for application of electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) methods. To this end model compounds are investigated as substrate analogues of lysozyme by x-ray diffraction and magnetic resonance methods.
Aromatic-linked mixed disaccharides corresponding in chemical structure to β(aryl)-4-0-(2-deoxy-2-acetamido- D-glucopyranosyl)-D-glucopyranoside are synthesised as substrate analogues of lysozyme to serve as phosphorescent triplet state spectroscopic probes. The stereochemistry and molecular structure of β(1-naphthyl)-D-glucopyranoside- 2',3',4',6'-tetra-0-acetate is determined by application of direct methods of structure determination and crystallographic least-squares refinement. The detailed molecular structure of this carbohydrate derivative is compared to that of other glucopyranosides, and the compatibility of the incorporation of the molecular structure of the naphthyl-glucopyranoside moiety only into aromatic-linked mixed disaccharide compounds for purposes of a triplet state spectroscopic probe of lysozyme action is tested by molecular model building studies.
ESR and optical detection of magnetic resonance techniques are employed to evaluate the electronic structure of the phosphorescent substrate analogue of lysozyme. incorporation of the structural unit of the β(1-naphthyl)-D-glucopyranoside chromophore into a variety of hydrophobic and hydroxylic environments causes readily detectable alterations in the zero-field splitting parameters of the phosphorescent triplet state of the naphthyl group. The zero-field splitting parameters and transitions of the naphthyl-glucopyranoside in these environments are readily distinguishable from those of the expected hydrolysis product α-naphthol. These results indicate that the triplet state properties of phosphorescent aromatic groups are sufficiently sensitive to environmental influences and can be employed as direct spectroscopic probes of enzyme action when suitably incorporated into synthetic substrate analogues.
The spin-label inhibitor of lysozyme β(2,2,6,6- tetramethyl-4-piperidinol-1-N-oxyl)-D-(2-deoxy-2-acetamido-glucopyranoside) is also synthesised as a spectroscopic probe of the active site. ESR and x-ray diffraction studies of single crystals of tetragonal hen egg white lysozyme show that the spin-label is bound in the enzyme crystal. The principal axis corresponding to the gzz-direction is identified by ESR. Application of ENDOR to this orientation results in three types of ENDOR lines arising from interaction of the nitroxide group with nearby hydrogen nuclei. This interaction is analysed in terms of an anisotropic dipole-dipole mechanism. interpretation of the ENDOR spectrum suggests that (i) a protein residue is within 3.7 Å of the nitroxide nucleus along the molecular z-axis of the nitroxide group and (ii) the nitroxide group is substantially exposed to the solvent channel or surface of the enzyme. The three-dimensional structure of the spin-label complex is determined by difference Fourier methods. The spin-label binds anomalously across subsites C and B of the lysozyme molecule in a configuration apparently stabilised by binding of the acetamido group in the specificity site in subsite C and by hydrophobic interactions between the methyl groups of the spin-label and Tryp-62 of the enzyme. The structural features of the complex determined by diffraction methods confirm the interpretation of the ENDOR spectrum. Furthermore, the ENDOR results indicate that specific proton sites can be detected in the active site regions of crystalline enzymes as a sensitive means to dilineate stereochemical configurations and structural binding relationships.
Since an enzymatically active form of crystalline lysozyme is prerequisite to further spectroscopic and structural study, the catalytic activity of crystalline human lysozyme is assessed. With use of a p-nitrophenyl-linked mixed disaccharide derivative, human lysozyme in orthorhombic crystals is demonstrated to be enzymatically active only when the crystals are equilibrated with a mixed cosolvent medium containing 2-methyl-pentan-2,4-diol. These solvent conditions have negligible effects on the crystal structure and are suitable for preserving the crystal structure at cryogenic temperatures at which ESR and ENDOR techniques must be generally applied.
To evaluate conditions appropriate for assaying the enzymatic activity of crystalline enzymes, the catalytic efficiency of crystalline enzymes is analysed mathematically as a function of the diffusion of substrate into crystals and of the kinetic parameters of the enzyme reaction in solution. Relationships are derived to demonstrate that crystal dimensions control the influence of diffusion in measuring the catalytic efficiency of crystalline enzymes and that the critical dimension for onset of rate-limiting diffusion can be predicted on the basis of measurable kinetic parameters of the enzyme reaction in solution and the invariant diffusion coefficient of the substrate. The catalytic efficiency of crystalline enzymes reported hitherto in the literature is analysed on the basis of these mathematical relationships. Except for the case of papain, it is concluded that the results of all other studies are reported for conditions of rate-limiting diffusion of substrate and are consequently inconclusive in comparing the catalytic activity of the enzyme in crystalline and solution states.
In addition, the appropriate chemical conditions are determined for generation and stabilisation of oxymyoglobin in single crystals with use of ferrous D(+)-tartrate in alkaline ammonium sulphate solutions under conditions suitable for high resolution x-ray structural studies.