Experimental Evidence for a Hydrophobic Active Center of Glutamine-Aspartic Transaminase.

It has been found that fluorescein dimercuric acetate acts as a potential inhibitor of transaminase. Although the holoenzyme was inhibited also by fluorescein isothiocyanate the latter fluorochrome was found to be a rather specific inhibitor of the apoenzyme. The interactions involved are fast, the induced effect reaching its maximum value in less than 1 min. The isolated fluorescein dimercuric acetate or fluorescein isothiocyanate and transaminase or apotransaminase complexes suggest that the fluorochromes reacts specifically with two active sulfhydryl or amino groups of the enzyme, respectively. The fluorescence intensity of the fluorochromes is substantially quenched during their interaction with transaminase and apotransaminase. A red shift in the absorption spectrum maximum from 498 nm to 512 nm was also observed when fluorescein dimercurie acetate solutions were titrated with transaminase or apotransaminase. The recorded shift for fluorescein isothiocyanate in the fluorescein-isothiocyanate · apotransaminase complex was from 493 nm to 502 nm, respectively. In contrast to the previous spectral changes a blue shift in the spectrum maximum from 498 nm to 478 nm was recorded during the stoichiometric interaction of fluorescein dimercurie acetate with cysteine. Comparative studies concerning the reactivity of fluorescein dimercuric acetate or fluorescein isothiocyanate with transaminase, apotransaminase and cysteine or lysine suggest that the alterations observed in the spectral and fluorescence properties of the fluorochromes during their interaction with transaminase and apotransaminase arose from their complexation with specific sulfhydryl groups of the enzyme. These groups should be located in a critical hydrophobic area of the transaminase molecule since their interaction abolishes the catalytic activity of the enzyme and alters the spectral and fluorescence properties of the fluorochromes in the same way as low polarity solvents. These sulfhydryl and amino groups might also play a functional role since they interact faster with fluorescein dimercuric acetate and fluorescein isothlocyanate than the sulfhydryl group of cysteine or the amino group of lysine. Quantitative parameters of the data obtained by conventional techniques and stopped-flow spectroscopy during the interaction of the enzyme with the fluorochromes are also given. [ABSTRACT FROM AUTHOR]