Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction.
1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(β) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(β) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.
Division of Medicinal Chemistry, College of Pharmacy, Department of Chemistry and Biochemistry, and Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, United States.
Christopher J
CJ
Thibodeaux
Hung-Wen
HW
Liu
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011549:Pseudomonas / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D011732:Pyridoxal Phosphate; C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases
Biochemistry. 1997 Dec 9;36(49):15477-88
9398277
Biochemistry. 1987 Jun 2;26(11):3027-32
3607008
Curr Opin Chem Biol. 2005 Oct;9(5):482-7
16125438
Proc Natl Acad Sci U S A. 1966 Apr;55(4):712-6
5219675
Biochemistry. 1992 Jun 30;31(25):5878-87
1610831
Biochemistry. 2002 Mar 26;41(12):4048-58
11900548
Biochemistry. 2003 Feb 25;42(7):2089-103
12590597
J Biol Chem. 2003 Oct 17;278(42):41069-76
12882962
Biochemistry. 1996 Apr 16;35(15):4776-83
8664267
Biochemistry. 2003 Apr 29;42(16):4658-68
12705829
J Am Chem Soc. 2004 Mar 31;126(12):3769-76
15038730
Biochemistry. 1981 Dec 22;20(26):7509-19
7326243
Chem Rev. 2006 Aug;106(8):3236-51
16895326
Biochemistry. 1992 Sep 15;31(36):8516-22
1390637
J Biol Chem. 2009 Jan 2;284(1):207-217
18955494
Methods Enzymol. 1982;87:390-405
7176923
Plant Cell. 1991 Nov;3(11):1187-93
1821764
Biochemistry. 2006 Nov 21;45(46):13889-98
17105207
Biochemistry. 1993 Jan 26;32(3):812-8
8422386
Biochemistry. 2002 Mar 19;41(11):3836-42
11888303
Anal Biochem. 1976 May 7;72:248-54
942051
Biochemistry. 1990 Jan 16;29(2):442-51
2405904
Structure. 2000 Feb 15;8(2):185-95
10673437
Arch Biochem Biophys. 2005 Jan 1;433(1):2-12
15581561
Biochemistry. 1987 Jun 16;26(12):3417-25
3651390
Biochemistry. 1998 Mar 10;37(10):3509-17
9521672
Angew Chem Int Ed Engl. 2004 Jun 28;43(26):3425-9
15221829
J Biol Chem. 2000 Nov 3;275(44):34557-65
10938279
J Mol Biol. 1995 Nov 10;253(5):799-812
7473753
J Mol Biol. 1993 Dec 20;234(4):1218-29
8263922
Biochemistry. 2009 Jun 23;48(24):5510-7
19435313
J Am Chem Soc. 1970 Jul 15;92(14):4435-40
5428387
Annu Rev Biochem. 2004;73:383-415
15189147
J Biol Chem. 1991 May 25;266(15):9374-81
2033039
Biochemistry. 1984 Mar;23(6):1275-82
11491129
Methods Enzymol. 1982;87:551-606
6294457
Biochemistry. 1997 Apr 15;36(15):4558-67
9109665
Biochemistry. 1982 Mar 16;21(6):1302-7
7074086
Biochemistry. 1984 Nov 6;23(23):5471-8
6391543
Biochemistry. 2004 Feb 10;43(5):1193-203
14756555
Arch Biochem Biophys. 2001 Apr 15;388(2):267-75
11368164
Biochemistry. 1975 Jun 17;14(12):2644-51
1148173
Arch Biochem Biophys. 2005 Jan 1;433(1):279-87
15581583
Biochemistry. 1999 Jan 5;38(1):311-20
9890912
Biochemistry. 2004 Oct 26;43(42):13328-39
15491139
Biochemistry. 1998 Oct 20;37(42):14943-7
9778371
Biochemistry. 2005 Dec 6;44(48):15930-6
16313196