Structure-activity relationship on fungal laccase from Rigidoporus lignosus: a Fourier-transform infrared spectroscopic study

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C

Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related