Comparison of amino acid sequence and thermostability of tyrosinase from three wild type strains of Neurospora crassa.

The thermostability of tyrosinase from three wild type strains of Neurospora crassa has been investigated. For this purpose a sequence comparison of two thermostable and one thermolabile tyrosinase isoenzyme was carried out. It revealed that at position 201 the thermostable enzyme forms share an aspartate residue in contrast to an asparagine residue in the thermolabile form. In addition, one of the thermostable isoenzymes displays five other substitutions. Since the relative stability of the thermostable forms as compared to the thermolabile one decreases with increasing ionic strength, the common aspartate residue is thought to bring about the additional stability of the thermostable isoenzymes by forming a salt bridge between aspartate 201 and a positively charged group of the protein. The strong pH-dependency of the thermostability with an apparent pKA of 6.6 indicates a histidinium side chain as the most likely ionic group to be involved in the salt bridge. This conjecture is also supported by measurements of the stability towards the chaotropic agent guanidinium chloride. The difference of the free energy change of denaturation delta GDH2O between the apoenzymes of a thermostable and a thermolabile isoenzyme was calculated as 2.5 kcal mol-1. Furthermore, it was shown that the copper ions of the native and the cobalt ions of Co(II)-substituted tyrosinase strongly enhance the stability of the protein as compared to its apoform.