GENETIC-ENGINEERING APPROACHES TO ENZYME DESIGN AND MECHANISM

Aspartate aminotransferase (AATase) and aminocyclopropane carboxylate synthase (ACC synthase) are pyridoxal phosphate (PLP)-dependent enzyme s whose common junction of mechanistic divergence is after the formati on of a C-alpha carbanion from the amino acid substrate bound to PLP a s a Schiff base (aldimine). AATase catalyzes the reversible interconve rsion of alpha-amino acids and alpha-keto acids, while ACC synthase ef fects the irreversible decomposition of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylate (ACC) and 5'-methylthioadenosine (MT A). ACC is subsequently converted to ethylene, the plant ripening and senescence hormone, by ACC oxidase, the next enzyme in the pathway. AA Tase and ACC synthase exhibit many similar phenomenological characteri stics that result from different detailed mechanistic origins. The k(c at)/K-M versus pH profiles for both enzymes are similar (AATase, acidi c pK(a) = 6.9, basic pK(a) = 9.6; ACC synthase, acidic pK(a) = 7.5, ba sic pK(a) = 8.9); however the acidic pK(a) of AATase reflects the ioni zation of an enzyme proton from the internal Schiff base, and the basi c one is that of the alpha-amino group of the substrate, while the opp osite situation obtains for ACC synthase, i.e. the apparent pK(a) of 7 .4 is due to the alpha-amino group of SAM, whereas that of 9 reflects the Schiff base pK(a). The mechanistic imperative underlying this reve rsal is dictated by the reaction mechanism and the low pK(a) of the al pha-amino group of SAM. The low pK(a) of SAM requires that the enzyme pK(a) be moved upward in order to have sufficient quantities of the re acting species at neutral pH. It is shown by viscosity variation exper iments with wildtype and active site mutant controls of both enzymes t hat the reaction of SAM with ACC synthase is 100% diffusion controlled (k(cat)/K-M = 1.2 x 10(6) l mol(-1) s(-1)) while the corresponding re action for the combination of L-aspartate with AATase is insensitive t o viscosity, and is therefore chemically not diffusion limited. Tyr225 (AATase) or Tyr233 (ACC synthase) forms a hydrogen bond with the PLP in both enzymes, but that formed with the former enzyme is stronger an d accounts for the lower pK(a) of the Schiff base. (C) 1998 John Wiley & Sons, Ltd.