Probing intra vs interchain kinetic preferences of L-Thr acylation on dimeric VibF with mass spectrometry
Leslie M Hicks 1, Carl J Balibar 2, Christopher T Walsh 2, Neil L Kelleher 1 and Nathan J Hillson 3*
1 University of Illinois at Urbana-Champaign
2 Harvard Medical School
3 Stanford University School of Medicine
* To whom correspondence should be addressed. E-mail: hillson{at}gmail.com.
Submitted on March 10, 2006
Revised on May 26, 2006
Accepted on 19 June 2006
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Abstract |
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We present a method to probe intra- and interchain activities within dimeric non-ribosomal peptide synthetases (NRPSs). Utilizing domain inactivation and analytical mass mutants in conjunction with rapid quench, mass spectrometry, and a probabilistic kinetic model, we have elucidated the pre-steady state intra- and interchain rates and the corresponding flux of the acylation of L-Thr onto VibF. While the intra rate is significantly faster than the inter rate, the data are most consistent with an even flux of covalent substrate loading where neither pathway dominates. These pre-steady state results confirm previous steady-state in vitro mutant complementation studies of VibF. Extension of this methodology to other dimeric NRPSs, and to the related fatty acid and polyketide synthases, will further our biophysical understanding of their acyl-intermediate-processing pathways.
Key Words:
analytical mass mutant, fatty acid, non-ribosomal peptide, polyketide, probabilistic kinetic model, rapid quench
