ISSN:
0894-3230
Keywords:
enzyme design
;
enzyme mechanism
;
genetic engineering
;
Chemistry
;
Theoretical, Physical and Computational Chemistry
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
,
Physics
Notes:
Aspartate aminotransferase (AATase) and aminocyclopropane carboxylate synthase (ACC synthase) are pyridoxal phosphate (PLP)-dependent enzymes whose common junction of mechanistic divergence is after the formation of a Cα carbanion from the amino acid substrate bound to PLP as a Schiff base (aldimine). AATase catalyzes the reversible interconversion of α-amino acids and α-keto acids, while ACC synthase effects the irreversible decomposition of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylate (ACC) and 5′-methylthioadenosine (MTA). ACC is subsequently converted to ethylene, the plant ripening and senescence hormone, by ACC oxidase, the next enzyme in the pathway. AATase and ACC synthase exhibit many similar phenomenological characteristics that result from different detailed mechanistic origins. The kcat/KM versus pH profiles for both enzymes are similar (AATase, acidic pKa = 6.9, basic pKa = 9.6; ACC synthase, acidic pKa = 7.5, basic pKa = 8.9); however the acidic pKa of AATase reflects the ionization of an enzyme proton from the internal Schiff base, and the basic one is that of the α-amino group of the substrate, while the opposite situation obtains for ACC synthase, i.e. the apparent pKa of 7.4 is due to the α-amino group of SAM, whereas that of 9 reflects the Schiff base pKa. The mechanistic imperative underlying this reversal is dictated by the reaction mechanism and the low pKa of the α-amino group of SAM. The low pKa of SAM requires that the enzyme pKa be moved upward in order to have sufficient quantities of the reacting species at neutral pH. It is shown by viscosity variation experiments with wild-type and active site mutant controls of both enzymes that the reaction of SAM with ACC synthase is 100% diffusion controlled (kcat/KM = 1.2 × 106 l mol-1 s-1) while the corresponding reaction for the combination of L-aspartate with AATase is insensitive to 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 and accounts for the lower pKa of the Schiff base. © 1998 John Wiley & Sons, Ltd.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
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