Stabilization of Proteases by an Engineered Disulfide Bond

Oligonucleotide-directed site-specific mutagenesis is a powerful technique for the study of proteins and has brought about great progress in research on the structure and function of proteins. This technique makes it possible in theory to obtain any mutant enzyme desired. One of the goals of such protein engineering is to make the proteins more stable. 

The disulfide bond differs from other types of interactions in folded proteins, such as hydrogen bonds and hydrophobic, electrostatic and van der Waals interactions. The disulfide bond is a covalent bond that is able to significantly stabilize folded conformations by 2-5 kcal/ mol for each disulfide.11 The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide.21 On the other hand, another view is that the disulfide bond destabilizes folded structures entropically, but stabilizes them enthalpically to a greater extent.31 

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

Natural thermostable enzymes are expected to be a good model for engineering stabilization of proteins. Study of thermostable enzymes from thermophilic microorganisms has revealed that the hydrophobicity of hydrophobic core inside the protein molecule and the electrostatic interactions of amino acid residues within the folded protein seem to be the cause of their stability. The enzymes from thermophilic microorganisms known so far do not contain the disulfide bond, but aqualysin I. Aqualysin I is an extracellular protease while the others are intracellular enzymes. 

As for subtilisin BPN, the first attempt at molecular modeling of disulfide mutants was performed with computer graphics using coordinates from the crystal structure of the enzyme,10-121 but increasing enzyme stability was unsuccessful. We have studied a thermostable subtilisin-type protease, aqualysin I, and the introduction site of a disulfide bond was chosen on structural homology between aqualysin I and subtilisin E. Here we describe a successful study to increase the stability of subtilisin E and others done for subtilisin enzymes. 

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