l-Phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis) : Explaining the kinetic effects of substrate modification by linear free-energy relationships

The action of phenylalanine ammonia-lyase [EC 4.3.1.5] on a series of para-substituted l-phenylalanines has been investigated. Multiple linear regression analysis has been used to relate the logs of k cat, K m , and k cat K m to substituent parameters for electron withdrawal, hydrophobic bonding, and size. The inhibitory action of the enantiomeric d-phenylalanines was also investigated. The results indicate that the rate-limiting step is not subsequent to the release of cinnamate from the enzyme. Explanations for the observed regression constants are discussed in terms of the influence of the substituent parameters on the dissociation constant for the bound substrate, the rate-limiting step, and intermediate steps such as the elimination process. The discussion utilizes a new theoretical treatment of the application of linear free-energy relationships to steady-state enzyme kinetics. It is shown that, in order to interpret structure-activity correlations in terms of rate and equilibrium constants for an unbranched catalytic sequence, a restricted model must apply. The reaction must have a single rate-limiting step so that quasi-equilibrium conditions prevail. In the QE-DS (quasi-equilibrium dominant-state) model, a single state of the enzyme-substrate complex is assumed to predominate. In the QE-FR (quasi-equilibrium fixed-ratio) model, changes in the enzyme substituents are assumed not to alter the ratio between the different forms of the enzyme-substrate complex prior to the rate-limiting step.