Kinetic and spectroscopic investigations of wild-type and mutant forms of apple 1-aminocyclopropane-1-carboxylate synthase

Two catalytically inactive mutant forms of 1 1-aminocyclopropane-1-carboxylate (ACC) synthase, Y85A and K273A, were mixed in low concentrations of guanidine hydrochloride (GdnHCl). About 15% of the wild-type activity was recovered (theoretical 25% for a binomial distribution), proving that the functional unit of the enzyme is a dimer, or theoretically, a higher order oligomer, The enzyme catalyzes the conversion of S-adenosyl-L-methionine (SAM) to ACC. The value of k(cat)/K-M is 1.2 x 10(6) M-1 s(-1) at pH 8.3. Viscosity variation experiments with glycerol and sucrose as viscosogenic reagents showed that this reaction is nearly 100% diffusion controlled. The sensitivity to viscosity for the corresponding reaction of the less reactive Y233F mutant is much reduced, thus the latter reaction serves as a control for that of the wild-type enzyme. The k(cat)/K-M vs pH profile for wild-type enzyme exhibits pK(a) values of 7.5 and 8.9. The former is assigned to the pK(a) of the alpha-amino group of SAM, while the latter corresponds to the independently determined spectrophotometric pK(a) of the internal aldimine, The k(cat) vs pH profile exhibits similar pK(a)s, which means that the above pK(a) values are not perturbed in the Michaelis complex, The phenolic hydroxyl group of Tyr233 forms a hydrogen bond to the 3'-O- of PLP, The spectral and kinetic pK(a) (k(cat)/K-M) values of the Y233F mutant are not identical (spectral 10.2, kinetic 8.7). A model that accounts quantitatively for these data posits two parallel pathways to the external aldimine for this mutant, the minor one has the alpha-amino group free base form of SAM reacting with the protonated imine form of the enzyme with k(cat)/K-M approximate to 6.0 x 10(3) M-1 s(-1), while the major pathway involves reaction of the aldehyde form of PLP with SAM with k(cat)/K-M approximate to 7.0 x 10(5) M-1 s(-1). The spectral pK(a) is defined only by the less reactive species.