title: Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase.
abstract: Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.
authors.affiliation: Department of Chemistry, University at Buffalo , Buffalo, New York 14260, United States.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhai
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.forename: Rik K
authors.initials: RK
authors.lastname: Wierenga
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Loria
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002645:Chickens; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D017905:Phosphites / Q000494:pharmacology; D011522:Protons; D011817:Rabbits; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000201:enzymology
chemical_list: D017905:Phosphites; D011522:Protons; D014305:Triose-Phosphate Isomerase; D000079:Acetaldehyde; C010972:glycolaldehyde
references.citation: Nature. 1970 Jul 11;227(5254):180-1
references.pmid: 5428407
references.citation: J Am Chem Soc. 2006 Jun 28;128(25):8110-1
references.pmid: 16787055
references.citation: Biochemistry. 2013 Jan 22;52(3):537-46
references.pmid: 23276261
references.citation: Biochemistry. 2012 Apr 3;51(13):2652-61
references.pmid: 22409228
references.citation: Nature. 1970 Jul 11;227(5254):181
references.pmid: 5428408
references.citation: J Biol Chem. 2003 Mar 14;278(11):9544-51
references.pmid: 12522213
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. 1992 Sep 15;31(36):8488-94
references.pmid: 1390633
references.citation: Biochemistry. 1994 Mar 15;33(10):2824-9
references.pmid: 7907502
references.citation: Biochemistry. 2013 Apr 2;52(13):2302-8
references.pmid: 23473304
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):50-5
references.pmid: 12509510
references.citation: J Am Chem Soc. 2003 Jul 30;125(30):8968-9
references.pmid: 15369325
references.citation: Biochemistry. 1991 Mar 26;30(12):3011-9
references.pmid: 2007138
references.citation: Biochem J. 1972 Sep;129(2):301-10
references.pmid: 4643318
references.citation: Biochemistry. 2008 Jul 29;47(30):7785-7
references.pmid: 18598058
references.citation: J Am Chem Soc. 2010 Sep 29;132(38):13525-32
references.pmid: 20822141
references.citation: Biochemistry. 2010 Sep 7;49(35):7704-8
references.pmid: 20687575
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: 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. 1994 Mar 15;33(10):2830-7
references.pmid: 8130195
references.citation: Biochemistry. 2004 Sep 14;43(36):11436-45
references.pmid: 15350130
references.citation: Biochemistry. 2009 Jun 2;48(21):4548-56
references.pmid: 19348462
references.citation: Biochemistry. 1992 Sep 15;31(36):8482-7
references.pmid: 1390632
references.citation: J Am Chem Soc. 1970 Apr 8;92(7):2170-2
references.pmid: 5461608
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: Biochemistry. 1976 Sep 7;15(18):4006-17
references.pmid: 963019
references.citation: Biochem J. 1972 Sep;129(2):311-20
references.pmid: 4643319
references.citation: J Am Chem Soc. 2008 Feb 6;130(5):1574-5
references.pmid: 18186641
references.citation: Proteins. 2010 Jun;78(8):1878-88
references.pmid: 20235230
references.citation: Protein Eng Des Sel. 2004 Apr;17(4):375-82
references.pmid: 15166315
references.citation: J Mol Biol. 2001 Apr 6;307(4):1103-12
references.pmid: 11286559
references.citation: Biochemistry. 2011 Jun 28;50(25):5767-79
references.pmid: 21553855
references.citation: Biochemistry. 2010 Feb 9;49(5):824-6
references.pmid: 20050635
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3784-8
references.pmid: 12824418
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: Biochemistry. 1975 Dec 2;14(24):5274-9
references.pmid: 47
references.citation: Biochemistry. 1990 Apr 3;29(13):3186-94
references.pmid: 2185832
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. 1988 Aug 9;27(16):5948-60
references.pmid: 2847777
references.citation: Biochemistry. 2012 Jun 12;51(23):4630-2
references.pmid: 22620855
references.citation: Biochemistry. 2009 Jun 23;48(24):5769-78
references.pmid: 19425580
references.citation: Biochemistry. 1999 Aug 31;38(35):11474-81
references.pmid: 10471299
references.citation: Eur J Biochem. 1986 Jun 2;157(2):441-53
references.pmid: 2940090
references.citation: Biochemistry. 1991 Jul 16;30(28):6948-56
references.pmid: 2069953
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. 1969 Aug 16;223(5207):704-5
references.pmid: 4979456
references.citation: Biochemistry. 2008 Aug 5;47(31):7983-5
references.pmid: 18620424
references.citation: J Mol Biol. 1992 Apr 20;224(4):1115-26
references.pmid: 1569570
references.citation: Curr Opin Chem Biol. 2001 Dec;5(6):626-33
references.pmid: 11738171
references.citation: Proteins. 1993 Aug;16(4):311-26
references.pmid: 8356028
references.citation: Biochemistry. 2010 Jun 29;49(25):5377-89
references.pmid: 20481463
references.citation: Proteins. 2006 Aug 15;64(3):700-10
references.pmid: 16741995
references.citation: Eur J Biochem. 1993 Feb 1;211(3):703-10
references.pmid: 8436128
references.citation: J Am Chem Soc. 2013 Apr 24;135(16):5978-81
references.pmid: 23560625