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Michael addition is an important reaction to form C–C bonds. Different hydrolases (e.g., lipases, proteases, and D-aminoacylase) have been reported to catalyze C–C forming reactions, but the reaction mechanism is not entirely clear. This study examined several model Michael reactions catalyzed by lipases and amino acids in various solvents, and found that “‘water-like”’ functionalized ionic liquids (ILs) increased the reaction yield to 35-55% from 30% in triglyme and 17% in [BMIM][Tf2N]. Interestingly, tertiary amides as solvents remarkably increased the reaction yield (to up to 65–85%) and enantioselectivity (up to 71–84% ee) when catalyzed by porcine pancreatic lipase (PPL). Our experimental, spectroscopic, and computational studies discovered that the lipase catalysis can be attributed to basic amino acid residues as the catalysts to promote Michael addition, especially when tertiary amide solvents partially unfold the protein and expose its basic amino acid residues.
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The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism
Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ,we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.
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- Award ID(s):
- 1856617
- PAR ID:
- 10496023
- Publisher / Repository:
- Elsevier Inc
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 299
- Issue:
- 1
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 102796
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.jbc.2022.102796
