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Title: An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.
Authors:
 [1] ;  [1] ;  [1] ;  [2] ;  [1] ;  [1] ;  [3] ;  [2] ;  [4] ;  [1]
  1. Mayo Clinic, Jacksonville, Florida (United States). College of Medicine, Dept. of Cancer Biology
  2. Ben-Gurion Univ. of the Negev, Beer-Sheva (Israel). Dept. of Biotechnology Engineering and the National Inst. of Biotechnology
  3. Brookhaven National Lab. (BNL), Upton, NY (United States). Photon Sciences Directorate
  4. Mayo Clinic, Jacksonville, Florida (United States). College of Medicine, Dept. of Neuroscience
Publication Date:
Report Number(s):
BNL-113701-2017-JA
Journal ID: ISSN 0021-9258
Grant/Contract Number:
SC0012704
Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 51; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Protein dynamic; protein conformation; serine protease; protease inhibitor; proteolysis; enzyme catalysis; protein structure; crystal structure; molecular dynamics; molecular modeling
OSTI Identifier:
1351724