title: Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.
abstract: Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.
authors.affiliation: Department of Physics, Emory University , Atlanta, Georgia 30322, United States.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Kurt
authors.initials: K
authors.lastname: Warncke
mesh_terms: D055162:Biocatalysis; D002244:Carbon / Q000737:chemistry*; D003035:Cobalt / Q000737:chemistry*; D003038:Cobamides / Q000378:metabolism*; D019277:Entropy; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011412:Propanolamines / Q000378:metabolism; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology
chemical_list: D003038:Cobamides; D011412:Propanolamines; D003035:Cobalt; D002244:Carbon; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide
references.citation: Chem Rev. 2006 May;106(5):1672-99
references.pmid: 16683749
references.citation: Biochemistry. 2003 Apr 22;42(15):4578-84
references.pmid: 12693955
references.citation: Angew Chem Int Ed Engl. 2012 Sep 10;51(37):9306-10
references.pmid: 22893270
references.citation: Biochemistry. 2005 Feb 22;44(7):2686-91
references.pmid: 15709782
references.citation: J Am Chem Soc. 2005 Jun 22;127(24):8856-64
references.pmid: 15954794
references.citation: Biochemistry. 2013 Feb 26;52(8):1419-28
references.pmid: 23374068
references.citation: Annu Rev Biochem. 2003;72:517-71
references.pmid: 12704087
references.citation: Angew Chem Int Ed Engl. 2011 Nov 11;50(46):10843-6
references.pmid: 21948289
references.citation: Protein Sci. 1996 Dec;5(12):2647-50
references.pmid: 8976574
references.citation: Nature. 2007 Jul 19;448(7151):325-9
references.pmid: 17637663
references.citation: Arch Biochem Biophys. 1999 Oct 1;370(1):138-41
references.pmid: 10496987
references.citation: J Am Chem Soc. 2006 Feb 1;128(4):1287-92
references.pmid: 16433547
references.citation: Proc Natl Acad Sci U S A. 1977 Jun;74(6):2236-40
references.pmid: 196283
references.citation: Biochemistry. 2007 Jan 23;46(3):883-9
references.pmid: 17223710
references.citation: Biochemistry. 2009 Jan 13;48(1):140-7
references.pmid: 19072291
references.citation: J Am Chem Soc. 2002 Nov 27;124(47):14054-65
references.pmid: 12440904
references.citation: Biochemistry. 1999 Sep 28;38(39):12681-9
references.pmid: 10504238
references.citation: Dalton Trans. 2006 Mar 7;(9):1123-33
references.pmid: 16482346
references.citation: Biochemistry. 1999 Jan 26;38(4):1234-42
references.pmid: 9930983
references.citation: Eur J Biochem. 1978 Jan 2;82(1):143-54
references.pmid: 620668
references.citation: J Am Chem Soc. 2005 Nov 30;127(47):16522-8
references.pmid: 16305240
references.citation: J Am Chem Soc. 2011 May 11;133(18):6968-77
references.pmid: 21491908
references.citation: Curr Opin Struct Biol. 2004 Feb;14(1):70-5
references.pmid: 15102452
references.citation: Biochemistry. 1999 Jan 26;38(4):1221-33
references.pmid: 9930982
references.citation: Biochemistry. 1997 Mar 25;36(12):3713-8
references.pmid: 9132024
references.citation: J Biol Chem. 1965 Dec;240(12):4669-74
references.pmid: 5846987
references.citation: J Phys Chem B. 2005 Sep 29;109(38):18146-52
references.pmid: 16853330
references.citation: Chem Rev. 2005 Jun;105(6):2075-149
references.pmid: 15941210
references.citation: Nucleic Acids Res. 1999 Jan 1;27(1):254-6
references.pmid: 9847194
references.citation: Biochemistry. 2000 Jul 11;39(27):7998-8006
references.pmid: 10891081
references.citation: J Am Chem Soc. 2004 Jul 7;126(26):8167-80
references.pmid: 15225058
references.citation: J Biol Chem. 1990 Jul 25;265(21):12462-6
references.pmid: 2197274
references.citation: Proc Natl Acad Sci U S A. 2007 Jun 5;104(23):9661-6
references.pmid: 17517615
references.citation: J Am Chem Soc. 2012 Feb 1;134(4):2367-77
references.pmid: 22239442
references.citation: Arch Biochem Biophys. 1992 Apr;294(1):50-4
references.pmid: 1550360
references.citation: Angew Chem Int Ed Engl. 2007;46(6):980-3
references.pmid: 17171745
references.citation: J Am Chem Soc. 2008 Apr 9;130(14):4846-58
references.pmid: 18341340
references.citation: Biophys J. 1987 Dec;52(6):1083-5
references.pmid: 3427197
references.citation: Curr Opin Struct Biol. 2013 Feb;23(1):75-81
references.pmid: 23246280
references.citation: Proteins. 2006 Aug 1;64(2):308-19
references.pmid: 16688781
references.citation: Biopolymers. 1970;9(10):1125-227
references.pmid: 4918636
references.citation: Biochemistry. 2000 Oct 3;39(39):12069-75
references.pmid: 11009622
references.citation: J Biol Chem. 2010 Aug 20;285(34):26484-93
references.pmid: 20519496
references.citation: J Biol Chem. 1974 Jul 25;249(14):4537-44
references.pmid: 4367219
references.citation: Biochemistry. 1998 Aug 25;37(34):11864-72
references.pmid: 9718309
references.citation: CRC Crit Rev Biochem. 1982;13(4):385-428
references.pmid: 6759038
references.citation: Chem Rev. 2003 Jun;103(6):2095-127
references.pmid: 12797825
references.citation: J Am Chem Soc. 2012 Jan 25;134(3):1591-9
references.pmid: 22229941
references.citation: Proteins. 2010 May 1;78(6):1339-75
references.pmid: 20099310