Dynamics and Thermodynamics of Genetically Engineered Enzymes

The possible effects of site-directed mutagenesis on the dynamic and thermodynamic properties of enzymatics have been studied using yeast phosphoglycerate kinase (PGK) as a test system. A variety of physico-chemical techniques have been used, including: isothermal and differential scanning microcalorimetry, fluorescence and fluorescence relaxation kinetics, Raman spectroscopy and depolarised Rayleigh scattering. The latter is a new technique, developed here, which shows promise in the study of dynamic properties of biological macromolecules. Wild-type yeast PGK and the His388→Gin mutant, a mutant thought to induce significant changes in interaction in the "hinge" region of PGK, were compared by all techniques. Both forms of the enzyme were prepared in high yield by multicopy plasmid techniques, using plasmids supplied by Dr. Fothergill-Gilmore (Edinburgh University). Isothermal microcalorimetry was used to measure the thermodynamic parameters (Kdis, DeltaH, DeltaS) for binding of the three readily available substrates (ATP, ADP, 3-phosphoglycerate) to the enzyme. Although there were small differences in dissociation constants between the wild-type and mutant proteins, consistent with Km differences observed by kinetic methods, there were no major differences in enthalpies of binding. In the case of 3PG binding, however, there were significant differences with preliminary enthalpic data for the wild-type enzyme published by other workers. Enthalpic binding differences observed in various buffer systems are possibly due to a hitherto unrecognized protonation change associated with PGK-3PG binding. Differential scanning calorimetry, performed for us at Yale University by Prof. J. M. Sturtevant, showed major differences in thermal stability between mutant and wild-type PGK, with the mutant being generally less stable to thermal unfolding. But, unfortunately, the thermogram of both forms of the enzyme shows some anomalies which, so far, have prevented detailed thermodynamic analysis of the thermal transition process. Fluorescence emission, excitation and relaxation studies, performed in collaboration with Dr. D. T. F. Dryden (Newcastle University), shows significant differences between wild-type PGK and the His388→Gin mutant, though in neither case are these properties affected by ligand binding. Results indicate an interesting enhancement of fluorescence energy transfer between tryptophan residues in the mutant enzyme, possibly as a result of increased hinge flexibility in this mutant. A new technique of depolarized Rayleigh scattering from dilute aqueous solution has been developed and shows promise in the study of protein dynamic effects. This technique essentially probes the very low frequency (< 50 cm-1 ) region of Raman scattering where large scale, highly damped, anharmonic motions of macromolecules are thought to occur. Trial experiments using lysozyme as a test system show significant low-frequency spectral shifts on binding of the tri-N-acetylglucosamine inhibitor, consistent with a "stiffening" of the protein induced by ligand binding in the active site cleft. The routine and reproducible application of this technique to larger macromolecules has yet to be perfected, but interesting and suggestive preliminary data have been obtained for the mutant PGK. Ligand binding to the ATP domain induces global "stiffening" effects similar to those observed with lysozyme. But, in contrast, binding of 3PG on the opposing domain of this enzyme results in shifts to lower frequencies consistent with a significant "loosening" of the entire structure. No such effects have been detected for the wild-type enzyme. The general conclusion that may be drawn is that although short-range protein-substrate interactions in the active sites of PGK are relatively unaffected by this mutation, replacement of His388 by glutamine in a region remote from the active sites but in a key position to control interdomain motion, results in measurable differences in large-scale dynamic processes in this protein which may be related to overall function. Additional work (reported in Appendix D) includes an examination of the possible use of Surface Enhanced Raman Scattering (SERS) to study the vibrational spectra of proteins in dilute aqueous solution