Regulatory properties of pyruvate carboxylase from Aspergillus nidulans: evidence for the presence of a masked activator site [proceedings].

These analyses show that this cyclic A M P phosphodiesterase.contains at least 15ng of zinc per activity unit. The sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of fraction 18 suggests that the specific activity of pure enzyme is at least 104 units/mg, and the results of Fujimoto et al . (1974), obtained at 37"C, suggest it may be as high as 160 units/mg at 30°C. Thus 1 mol (65000g; Fujimoto et al . , 1974; Londesborough, 1974) contains about 1.6g-atoms of zinc. We are now trying t o make large enough amounts of pure enzyme t o permit accurate zinc analyses and a reliable estimate of the A&. When 1,lO-phenanthroline was added to reaction mixtures it inhibited the enzyme in a simple competitive fashion (Ki = 4 8 0 , ~ ~ ) . Attempts to prevent this inhibition by addition of metals, such as Zn, Ni and Co, which are strongly chelated by 1,lO-phenanthroline, were complicated by the strong inhibition exerted by these metals themselves (Fujimoto et al . , 1974). The inhibition by 1-3m~-l,10-phenanthroline was decreased by small amounts of NiS04, but the protection was never complete. Maximum protection (an approximate doubling of the reaction rate) was reached when the concentration of NiS04 was a third of that of the 1 ,lo-phenanthroline, corresponding to formation of Ni(OP)3. The enzyme was not inactivated by preincubation with 5m~-l , lO-phenanthroline for 2 h a t room temperature before assay of 50-fold diluted samples. By contrast, the enzyme was inactivated by preincubation with 8-hydroxyquinoline. The extent of inactivation depended on the concentration of 8-hydroxyquinoline, and the temperature and duration of the incubation. At 30°C, 40 %of the activity was lost in 1 min, and the remainder in 90min at 5 m~-8-hydroxyquinoline, and 50 % was lost in 160min at 0.5 m ~ 8 hydroxyquinoline. This irreversible inactivation was prevented by addition of 1 mg of bovine albumin/ml, so that an instantaneous inhibition by 8-hydroxyquinoline could be studied in reaction mixtures containing 1 mg of bovine albumin/ml. Under these conditions, 8-hydroxyquinoline was a simple competitive inhibitor (K, = 1.1 mM). In the absence of albumin, mixed inhibition was observed, and results indicated that significant irreversible inactivation occurred during the time (1 min) required to measure the reaction rate. Inhibition by mercaptoethanol was studied by the spectrophotometric assay, and was mixed (KF = 0 . 4 m ~ , KFs = 1 . 6 m ~ ) . Inhibition by mercaptoethylamine was similar, but non-linear. At 0 .5m~-cycl ic AMP, ~ O ~ M K C N caused 70% inhibition (t.1.c. assay), but NaN3 had no effect. This is consistent with the much smaller stability constants of the complexes of Znz+ with N3than with CN-. Inhibition by mercaptoethylamine and CNis not likely to be caused by binding to a hydrophobic site (rather than a metal), as might be the case for 1 ,lo-phenanthroline and 8-hydroxyquinoline. Further, the small size of the CNligand argues that if its binding site is not actually a part of the enzyme's catalytic site, it must in any case be very close.