Tentative rules for increasing the thermostability of enzymes by protein engineering

The advent of recombinant DNA techniques provides protein chemistry with a powerful tool for designing and modifying, via site-directed mutagenesis, the physicochemical characteristics of enzymes. Among these characteristics is thermostability. Since site-directed mutagenesis has to be applied to replace as few amino acids as possible, it is necessary to know the rules that govern protein thermostability. To gain insight into these rules, we have performed the analysis of replacements between mesostable/thermostable counterparts of isoenzymes, based on a table of replacements for tyrosinases of Neurospora crassa. Upon prediction of the secondary structure and hydropathic profiles, we found that replacements are conservative in type and length of secondary structure and that they occur preferentially in external regions of the proteins. Some tentative rules for applying site-directed mutagenesis to proteins are proposed and discussed.