Stability of the complex formed between French bean (Phaseolus vulgaris) phenylalanine ammonia-lyase and its transition-state analog.
Phenylalanine ammonia-lyase forms trans-cinnamate from L-phenylalanine, and thus stands at a gateway to secondary metabolism in higher plants. L-alpha-Amino-oxy-beta-phenylpropanoic acid (L-AOPP), a very effective competitive inhibitor of this enzyme, is most probably a transition-state analog for the elimination reaction. A preparation of phenylalanine ammonia-lyase (PAL), obtained from diluted suspension cultures of French bean cells, was used to investigate the binding of this compound in vitro. After extensive dialysis, the inhibitor remained tightly bound to the enzyme unless both an increased temperature and L-phenylalanine were provided, when the spectrophotometer trace of enzyme activity gradually approached linearity. Under such optimal catalytic conditions (37 degrees C; 25 mM L-phenylalanine; pH 8.8), dissociation of the enzyme-ligand complex took place with a half-time of approx 10 min. (This is much longer than reported for the enzyme from maize.) The consequences of these findings are discussed for investigations where L-AOPP is applied in vivo. These experiments have shown that the irreversible binding of the transition-state analog under appropriate conditions (0-4 degrees C, no L-phenylalanine) gave continued protection against attack on the enzyme by an excess of borohydride. By titrating the enzyme with increasing concentrations of analog and measuring the degree of protection afforded, the active-site concentration has been estimated. The turnover number (kcat = 0.8 s-1) given by this novel approach is of the same order of magnitude as previously reported from extensive purification of enzyme from other species.
D000642:Ammonia-Lyases / Q000378:metabolism*; D001894:Borohydrides / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D008433:Mathematics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*
D001894:Borohydrides; C019145:alpha-aminooxy-beta-phenylpropionic acid; D010649:Phenylalanine; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase