title: Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.
abstract: The mechanistic imperatives for catalysis of deprotonation of α-carbonyl carbon by triosephosphate isomerase (TIM) are discussed. There is a strong imperative to reduce the large thermodynamic barrier for deprotonation of carbon to form an enediolate reaction intermediate; and, a strong imperative for specificity in the expression of the intrinsic phosphodianion binding energy at the transition state for the enzyme-catalyzed reaction. Binding energies of 2 and 6 kcal/mol, respectively, have been determined for formation of phosphite dianion complexes to TIM and to the transition state for TIM-catalyzed deprotonation of the truncated substrate glycolaldehyde [T. L. Amyes, J. P. Richard,Biochemistry2007,46, 5841]. We propose that the phosphite dianion binding energy, which is specifically expressed at the transition state complex, is utilized to stabilize a rare catalytically active loop-closed form of TIM. The results of experiments to probe the role of the side chains of Ile172 and Leu232 in activating the loop-closed form of TIM for catalysis of substrate deprotonation are discussed. Evidence is presented that the hydrophobic side chain of Ile172 assists in activating TIM for catalysis of substrate deprotonation through an enhancement of the basicity of the carboxylate side-chain of Glu167. Our experiments link the two imperatives for TIM-catalyzed deprotonation of carbon by providing evidence that the phosphodianion binding energy is utilized to drive an enzyme conformational change, which results in a reduction in the thermodynamic barrier to deprotonation of the carbon acid substrate at TIM compared with the barrier for deprotonation in water. The effects of a P168A mutation on the kinetic parameters for the reactions of whole and truncated substrates are discussed.
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhai
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260, USA.
authors.forename: M Merced
authors.initials: MM
authors.lastname: Malabanan
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260, USA.
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):50-5
references.pmid: 12509510
references.citation: J Mol Biol. 2001 Jun 29;310(1):271-80
references.pmid: 11419952
references.citation: Biochemistry. 1976 Sep 7;15(18):4006-17
references.pmid: 963019
references.citation: Biochemistry. 1975 Dec 2;14(24):5274-9
references.pmid: 47
references.citation: Biochemistry. 2013 Mar 26;52(12):2021-35
references.pmid: 23327224
references.citation: Philos Trans R Soc Lond B Biol Sci. 1991 May 29;332(1263):115-21
references.pmid: 1678530
references.citation: Biochemistry. 2009 Jun 23;48(24):5769-78
references.pmid: 19425580
references.citation: Bioorg Chem. 2004 Oct;32(5):354-66
references.pmid: 15381401
references.citation: J Am Chem Soc. 2013 Apr 24;135(16):5978-81
references.pmid: 23560625
references.citation: J Am Chem Soc. 2010 Sep 29;132(38):13525-32
references.pmid: 20822141
references.citation: J Am Chem Soc. 2001 Nov 14;123(45):11325-6
references.pmid: 11697989
references.citation: Proc Natl Acad Sci U S A. 1981 Jul;78(7):4046-50
references.pmid: 16593049
references.citation: Biochemistry. 2007 May 15;46(19):5841-54
references.pmid: 17444661
references.citation: Biochemistry. 2013 Aug 27;52(34):5928-40
references.pmid: 23909928
references.citation: Curr Opin Struct Biol. 2010 Dec;20(6):702-10
references.pmid: 20951028
references.citation: J Am Chem Soc. 2011 Oct 19;133(41):16428-31
references.pmid: 21939233
references.citation: Biochemistry. 2009 Jun 2;48(21):4548-56
references.pmid: 19348462
references.citation: J Am Chem Soc. 2005 Nov 16;127(45):15708-9
references.pmid: 16277505
references.citation: Biochemistry. 2005 Feb 22;44(7):2610-21
references.pmid: 15709774
references.citation: J Am Chem Soc. 2012 Jun 20;134(24):10286-98
references.pmid: 22583393
references.citation: J Biol Chem. 1959 May;234(5):1007-10
references.pmid: 13654309
references.citation: Biochemistry. 2008 Apr 22;47(16):4575-82
references.pmid: 18376850
references.citation: J Am Chem Soc. 2008 Feb 6;130(5):1574-5
references.pmid: 18186641
references.citation: Acc Chem Res. 2001 Dec;34(12):938-45
references.pmid: 11747411
references.citation: Biochemistry. 1995 Jul 4;34(26):8309-19
references.pmid: 7599123
references.citation: Protein Eng Des Sel. 2004 Apr;17(4):375-82
references.pmid: 15166315
references.citation: Biochemistry. 1991 Jun 18;30(24):5821-6
references.pmid: 2043623
references.citation: Biochemistry. 2011 Jun 28;50(25):5767-79
references.pmid: 21553855
references.citation: Biochem Biophys Res Commun. 1978 Jul 14;83(1):347-52
references.pmid: 29623
references.citation: Biochemistry. 1970 Aug 18;9(17):3404-7
references.pmid: 5535476
references.citation: Biochemistry. 1990 Jul 17;29(28):6619-25
references.pmid: 2204418
references.citation: Biochemistry. 2012 Apr 3;51(13):2652-61
references.pmid: 22409228
references.citation: J Biol Chem. 1998 Oct 9;273(41):26257-60
references.pmid: 9756847
references.citation: Biochemistry. 2013 Mar 26;52(12):2009-11
references.pmid: 23452047
references.citation: Biochemistry. 2007 May 22;46(20):6001-10
references.pmid: 17455914
references.citation: Biochem J. 1979 Jun 1;179(3):607-21
references.pmid: 38777
references.citation: Biochemistry. 2006 Dec 26;45(51):15483-94
references.pmid: 17176070
references.citation: Cell Mol Life Sci. 2010 Dec;67(23):3961-82
references.pmid: 20694739
references.citation: Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):159-71
references.pmid: 6115415
references.citation: Biochemistry. 2003 Mar 18;42(10):2941-51
references.pmid: 12627960
references.citation: Acc Chem Res. 2002 Jun;35(6):341-9
references.pmid: 12069618
references.citation: J Biol Chem. 2003 Mar 14;278(11):9544-51
references.pmid: 12522213
references.citation: Biochemistry. 2013 Mar 26;52(12):2050-67
references.pmid: 23488725
references.citation: Biochemistry. 2005 Feb 22;44(7):2622-31
references.pmid: 15709775
references.citation: Nature. 1991 Mar 14;350(6314):121-4
references.pmid: 2005961
references.citation: Biochemistry. 2012 Jun 12;51(23):4630-2
references.pmid: 22620855
references.citation: Adv Enzymol Relat Areas Mol Biol. 1975;43:219-410
references.pmid: 892
references.citation: J Am Chem Soc. 2011 May 4;133(17):6545-8
references.pmid: 21486036
references.citation: Nature. 1948 May 8;161(4097):707-9
references.pmid: 18860270
references.citation: J Am Chem Soc. 2011 Dec 21;133(50):20052-5
references.pmid: 22053826
references.citation: Biochemistry. 1996 Sep 24;35(38):12387-401
references.pmid: 8823174
references.citation: Biochemistry. 1998 Mar 31;37(13):4305-9
references.pmid: 9556344
references.citation: Curr Opin Chem Biol. 2001 Dec;5(6):626-33
references.pmid: 11738171
references.citation: J Biol Chem. 1998 Oct 16;273(42):27035-8
references.pmid: 9765214
references.citation: Biochemistry. 2010 Jun 29;49(25):5377-89
references.pmid: 20481463