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Genetic engineering approaches to enzyme design and mechanism
Author(s) -
Feng L.,
Li Y.,
Kirsch J. F.
Publication year - 1998
Publication title -
journal of physical organic chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.325
H-Index - 66
eISSN - 1099-1395
pISSN - 0894-3230
DOI - 10.1002/(sici)1099-1395(199808/09)11:8/9<536::aid-poc62>3.0.co;2-b
Subject(s) - chemistry , mechanism (biology) , biochemical engineering , protein engineering , enzyme , computational biology , combinatorial chemistry , biochemistry , epistemology , engineering , philosophy , biology
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 C α 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 k cat / K M versus pH profiles for both enzymes are similar (AATase, acidic p K a = 6.9, basic p K a = 9.6; ACC synthase, acidic p K a = 7.5, basic p K a = 8.9); however the acidic p K a 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 p K a of 7.4 is due to the α‐amino group of SAM, whereas that of 9 reflects the Schiff base p K a . The mechanistic imperative underlying this reversal is dictated by the reaction mechanism and the low p K a of the α‐amino group of SAM. The low p K a of SAM requires that the enzyme p K a 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 ( k cat / K M = 1.2 × 10 6 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 p K a of the Schiff base. © 1998 John Wiley & Sons, Ltd.