Tryptophan-activating enzyme purification

Selenoprotein A is remarkably heat stable, as seen by the loss of only 20% of activity on boiling at pH 8.0 for lOmin (Thrner and Stadtman 1973). Although selenoprotein A contains one tyrosine and no tryptophan residues, it contains six phenylalanine residues and thus has an unusual absorbance spectrum (Cone et al. 1977). Upon reduction, a unique absorption peak emerges at 238 nm, presumably due to the ionized selenol of selenocysteine, which is not present in the oxidized enzyme. The activity of selenoprotein A was initially measured as its ability to complement fractions B and C for production of acetate from glycine, in the presence of reducing equivalents (e.g., dithiothreitol). Numerous purification schemes were adopted for isolation of selenoprotein A, all of which employed the use of an anion exchange column to exploit the strongly acidic character of the protein. 

Another effect of the heme cofactor is in the inactivation of the enzyme. When the bovine enzyme is mixed with hematin or hemoglobin, enzyme activity is lost rather rapidly. The rate of enzyme inactivation depends on the heme/enzyme ratio. As the amount of heme is raised beyond the equimolar quantity, the inactivation occurs faster. The half life of the enzyme is approximately 30 min when incubated with 5-fold excess of hematin or of hemoglobin at 24°C, pH 7.4. All the active heme compounds including the mangano analogue inactivate the enzyme. Both the cyclooxygenase and hydroperoxidase activities are lost in parallel [27]. A similar effect is observed with the ovine enzyme, and peroxide is presumed to participate in the heme-induced inactivation [39]. Such an enzyme-destroying action of heme hampers the purification and kinetic studies of the enzyme. Fortunately, however, the enzyme is protected from the heme-induced inactivation by the simultaneous presence of tryptophan, epinephrine, hydroquinone [27], phenol [27,39] and other compounds, all of which are cofactors for the hydroperoxidase reaction (see below). 

A purification and some properties of proteinase A from yeast are described. A specific macromolecular inhibitor of proteinase A from yeast cytosol has been isolated and shown to be a protein (molecular weight 7,700) consisting of a majority of polar amino acids. Proline, arginine, cysteine and tryptophan were not detected in the inhibitor. Possible biological functions of proteinase A and the proteinase A-inhibitor (and of other yeast proteinases and their inhibitors) in the following processes are discussed general protein turnover, catabolite inactivation of enzymes, enzyme degradation at starvation and at transition to spore formation, and activation of pre-enzymes and precursor proteins by limited proteolysis. 

Speedie (4S) has obtained a cell-free preparation from S. flocculus which catalyzes the formation of (31) from L-tryptophan and S-adenosyl-L methionine. The crude enzyme has been purified 2-fold by ammonium sulfate fractionation, and preliminary results with this preparation after dialysis indicated that pyridoxal phosphate is not required, but may cause some stimulation of enzyme activity. At this stage of purification tryptophan transaminase activity was also present, and it has not yet been possible to determine whether the true methylase substrate is an activated tryptophan, or indole pyruvic acid (33), as has been demonstrated to be the case in the biosynthesis of indolmycin (34) (46). 

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