The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.
trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R(G)) disappeared in the spectrum of the inactive alphaR8A mutant (deltaH = 7.48 ppm, deltaN = 89.6 ppm), thereby assigning these resonances to alphaArg-11NepsilonH, and alphaArg-8NepsilonH, respectively. (1)H-(15)N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoic acid (3-CBA), a competitive inhibitor (K(I)(slope) = 0.35 +/- 0.06 mM), resulted in progressive downfield shifts of the alphaArg-8Nepsilon resonance yielding a K(D) = 0.77 +/- 0.44 mM, comparable to the (K(I)(slope), suggestive of active site binding. Increasing the pH of free CaaD to 8.9 at 5 degrees C resulted in the disappearance of all nine Arg-NepsilonH resonances due to base-catalyzed NepsilonH exchange. Saturating the enzyme with 3-CBA (16 mM) induced the reappearance of two NepsilonH signals, those of alphaArg-8 and alphaArg-11, indicating that the binding of the substrate analogue 3-CBA selectively slows the NepsilonH exchange rates of these two arginine residues. The kinetic and NMR data thus indicate that betaPro-1 is the acid catalyst, alphaGlu-52 is a reasonable candidate for the general base, and alphaArg-8 and alphaArg-11 participate in substrate binding and in stabilizing the aci-carboxylate intermediate in a Michael addition mechanism.
Azurmendi
Hugo F
HF
Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Wang
Susan C
SC
Massiah
Michael A
MA
Poelarends
Gerrit J
GJ
Whitman
Christian P
CP
Mildvan
Albert S
AS
D000179:Acrylates / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004252:DNA Mutational Analysis; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011522:Protons; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D012997:Solvents
D000179:Acrylates; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D021122:Protein Subunits; D011522:Protons; D012997:Solvents; C055748:chlorovinylglycine; D001120:Arginine; D011392:Proline; D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase; D019746:Intramolecular Oxidoreductases; C048474:oxaloacetate tautomerase; D005998:Glycine