Investigation of the substrate binding sites of elastase
Studies of pancreatic elastase were undertaken to examine substrate binding and of human leukocyte elastase to try to gain insight into the disease, pulmonary emphysema. Porcine pancreatic elastase was inhibited by various inhibitors p-toluene sulphonyl fluoride, phenylmethane sulphonyl fluoride, 1-dimethylamino-naphthalene-S-sulphonyl fluoride or chloride and p-nitrophenyl-anthranilate. The enzyme was only fully inhibited by the first two inhibitors which were then treated under alkaline conditions to convert the serine-19S, -CH20H to -CH2 to form the anhydroelastase. The enzyme was shown to be totally inactive but X-ray diffraction data to 2.SA resolution from one preparation showed no changes at the active site. Further studies were carried out to modify the serine-19S to cysteine by thiolation of the inhibited enzyme and anhydroelastase but proved unsuccessful. Binding studies were carried out with the prepared hexapeptide substrate, H.Pro-Ala-Pro-Ala-Lys-Phe.OH and tetrapeptide inhibitors, Ac-Pro-Ala-Pro-Ala.OH (1), Ac-Pro-Ala-Pro-Alaninal (2) and TFA-Pro-Ala-Pro-Alaninal (3) using anhydroelastase and native elastase. Inhibition with inhibitors (2) and (3) showed TFA-peptide-aldehyde bound tighter than Ac-peptide-aldehyde with K for (2) - 6.1 llM and for (3) - 8.6 llM. A TFA-pepiide-chloromethYlketone was also examined. It showed anomalous binding with the TFA group bound to the active site region whilst the rest of the peptide bound in parallel mode with the main chain residues 214-216. Data on substrate (1) was collected but no binding around the active site region was observed. Small crystals of human leukocyte elastase were obtained by the technique of vapour diffusion, however sealed tube X-ray experiments and using the sychroton revealed no diffraction pattern~ Moreover, studies were done on the enzyme to remove the sugar content using various glycosidases of which almond emulsin showed some conclusive results. In addition, the crystal structures of two small molecules containing the heavy atoms ruthenium and platinium were solved by conventional techniques of X-ray crystallography.