Proteolytic Activity Study Of Thiol And Seleno-subtilisin

Subtilisin is a serine proteolytic enzyme which can not only hydrolysis peptide into amino acid but also can be used to catalyze the synthesis of peptides in organic solvents. Along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies.Mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature.Protein engineering of subtilisin commenced in the mid 1960s when the active site serine 221 was converted to cysteine through chemical modification by Neet work team.Cysteine221 subtilisin is catalytically wounded to the point that it will barely hydrolyze peptide bonds but turns out to be quite reactive with certain activated ester substrates. In 1990, Hilvert etc turned the hydroxy of the subtilisin active site Serine 221 into selenyl by chemically modified method.Such change make subtilisin almost lose its hydrolysis activity , but produce a new ability to simulate natural GPX antioxidant enzymes.There are some restriction of chemical modification methods when it was used in the protein engineering ,like the organic solvents may change the conformation or destroy the activity of the enzyme etc.Along with the development of the method of gene mutation, it was used in enzyme molecular modification more and more. We change the active site of subtilisin serine 221 into cysteine by site-directed mutagenesis methods and successfully expressed thiolsubtilisin and selenosubtilisn whose active site are cysteine and selenocysteine by Escherichia.coli expression system and auxotrophic expression system.A new method of the preparation of selenosubtilisin by biosynthensis was constructed.When a large amount of the exogenous gene is expressed in Escherichia.coli, the excessive nascent peptide chain are condensed down to form inclusion bodies before they could fold into the correct protein structure. Such phenomenon appeared in our expression, too. After cell disruption and highly centrifuged, the precipitation was dissolved in 6M guanidinium hydrochloride and it was purified under the condition of denaturation. After that we try to obtain our desired protein by the renaturation of inclusion bodies. Formation of inclusion bodies can protect the desired protein of exogenous gene from degradation of proteolytic enzyme produced by Escherichia coli and both the separation and the purification of inclusion bodies are simple due to its high density and large size. However, the renaturation of inclusion bodies has been a great problem for a long time. Subtilisin requires the amino-terminal propeptide of 77 amino acid residues for the formation of the active enzyme. The propeptide is cleaved upon completion of folding.Our purified protein was renatured by stepwise dialysis procedure against 10 mM Tris-HC1 (pH 7.0) containing0.5 M (NH4)2SO4, 1 mM CaCl2 and a variable amount of urea.and the protein sample was first dialyzed against 100 volumes of buffer containing 4 M urea at 4℃for 2 h. Then, the initial buffer was sequentially replaced every 2 h with buffer containing decreasing amounts of urea from 2 to l M and then to 0.5 M. Finally, urea was completely removed from the buffer.5mM DTT was added in the renaturation buffer of thiolsubtilisin to prevent the formationof mismatched disulfide bond. At each dialysis step, a 200μL sample was taken and analyzed by 15% SDS-PAGE.Using the above methods we successfully espressed wild type subtilisn, thiolsubtilisn and selenosubtilisn whose active site are respectively serine221, cysteine221 and selenocysteine221. The catalytic activity of the hydrolysis of p-nitrophenyl acetate by the three enzyme is measured and the kinetic parameters are obtained from the double-reciprocal plots of the initial velocity versus substrate concentration. Certain hydrogen bond network is formed among the catalytic triad of the active site of subtilisn during the hydrolysis of peptide or ester. Hydroxyl of serine transfers a proton to histidine when the substrate is near the active site pocket of subtilisn, then attacks the carbonyl group of substrate acting as a nucleophilic group. Therefore the hydrolysis ability of the enzyme depends on the proton transferred ability of the amino acid residue of the active site .As serine of the active site of subtilisin was mutated into cysteine and selenocysteine, it is the same way as the substitution of oxygen element of hydroxyl by sulphur element of sulfydryl and selenium element of hydrogen selenide.Oxygen , sulphur and selenium are in the same main group of the periodic table of elements, their chemical properties are similar, However, due to the increase of atomic radius from oxygen to selenium ,the loss of electron capacity enhance , therefore its corresponding enzymatic catalytic ability decreases gradually. Ir's proved that our theoretical speculation are correct according to our kinetics study of the enzymes.