Adenylosuccinase from muscle, liver and yeast is strongly inhibited by the substrate analogue adenylophosphonopropionate (N6-(DL-1-carboxy-2-phosphonoethyl)-adenosine 5'-monophosphate). The inhibition is freely reversible and of the competitive type, with apparent K1 values between 5.4 and 86 nM depending on the source of enzyme. Ratios of Km/K1 with adenylosuccinate as substrate fall in the range of 44 to 1350. Comparison of four carboxyl analogues of adenylosuccinate with the corresponding phosphonate analogues shows that the phosphonates are much better inhibitors. Adenylosuccinate analogues in which the beta-carboxyl is replaced by other functional groups are much poorer inhibitors. The exceptionally high affinity of adenylosuccinase for adenylophosphonopropionate appears to involve the dianion of the phosphonate group.
Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine-alpha-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine-pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.
Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle.
Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.
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