Abstract
Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.
Original language | English (US) |
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Pages (from-to) | 327-335 |
Number of pages | 9 |
Journal | Chemistry and Biology |
Volume | 11 |
Issue number | 3 |
DOIs | |
State | Published - Mar 2004 |
Funding
We acknowledge the generous support from the University of Illinois and an NSF Fellowship to L.M.H. N.L.K. and C.T.W. acknowledge the National Institutes of Health (GM 067725) and (GM 20011), respectively, and S.E.O. acknowledges an Irving Sigal postdoctoral fellowship (American Chemical Society).
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmacology
- Drug Discovery
- Clinical Biochemistry