Subunit interactions in enzyme catalysis. Kinetic analysis of subunit interactions in the enzyme L-phenylalanine ammonia-lyase.

Abstract

The effects of subunit interactions on the steady‐state rate of the reaction catalyzed by the enzymel‐phenylalanine ammonia‐lyase, have been investigated using the concepts of structural kinetics developed in a previous paper. Both theoretical and experimental methods have been proposed, which allow a choice among the possible kinetic models. These methods rest on the mathematical analysis of rate data obtained in the presence of substrate analogues, used as inhibitors.In the absence of inhibitor, the reciprocal plots exhibit a significant downward curvature. If a substrate analogue is present in the reaction medium, the plots can be straight lines, or can even exhibit an upward curvature. If vs and vI are the steady‐state rate in the absence and in the presence of inhibitor, respectively, the plots (vs/vI)‐1 versus inhibitor concentration are either straight lines if the inhibitor is d‐phenylalanine, or parabolas if the inhibitor is benzoic acid.The detailed analysis of these data as well as previous information obtained on the number of polypeptide chains, suggest that the enzyme is made up of two protomers (Mr 160X103), each protomer consisting of two. polypeptide chains: an α chain (Mr 75X103) and a β chain (Mr 85X103). The kinetic data indicate the existence of a hybrid conformation of the enzyme during substrate binding. Thus, phenylalanine ammonia‐lyase appears to follow neither the classical allosteric model of Monod, nor the simple sequential model of Koshland, but a partially‐concerted mechanism of subunit interactions. The binding of the substrate induces a conformation change in the corresponding subunit as well as the appearance of a new conformation of the unliganded protomer.When d‐phenylalanine or benzoic acid are used as inhibitors, no hybrid enzyme · substrate · inhibitor complex is formed. It thus appears that the new unliganded conformation, called A′, is still able to bind the substrate, l‐phenylalanine, but cannot form a complex with d‐phenyl‐alanine or benzoid acid.The behaviour of the free enzyme appears to be different with regard to D‐phenylalanine and benzoic acid. The unliganded phenylalanine ammonia‐lyasc can bind one molecule of d‐phenylalanine only, whereas it can bind two molecules of benzoic acid. It thus appears that the binding of d‐phenylalanine on a subunit, induces a conformation change of the unliganded subunit, the new conformation (A″) being unable to form a complex with any molecule of inhibitor. In the case of benzoic acid, on the other hand, the new conformation of the unliganded protomer is still able to bind an inhibitor molecule.