科研之友
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摘要
The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-L-alanine (OIA), with the natural substrate, L-tryptophan, and with a substrate S-ethyl-L-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme (%26quot;induced fit%26quot;) is realized in the case of mutant Y72F enzyme reaction with CIA. For the reaction of mutant enzyme with L-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates (%26quot;selected fit%26quot;). For the reaction with S-ethyl-L-cysteine the observed concentration dependence of k(obs) agrees with the realization of both kinetic schemes, the %26quot;selected fit%26quot; becoming predominant at lower concentrations of substrate, the %26quot;induced fit%26quot; at higher ones. In the reaction with S-ethyl-L-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic alpha,beta-elimination of ethylthiol from S-ethyl-L-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.
- 出版日期2014-10
