The Roles of Active-Site Residues in the Catalytic Mechanism of trans-3-Chloroacrylic Acid Dehalogenase:  A Kinetic, NMR, and Mutational Analysis

Abstract

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 c...

A persistent pesticide residue and the unusual catalytic proficiency of a dehalogenating enzyme

“…5), an unusually efficient enzyme that catalyzes a water-addition reaction with spontaneous heat and entropy of activation (⌬H ‡ ϭ 28.9 kcal͞ mol; T⌬H ‡ ϭ Ϫ6.8 kcal͞mol) (12) that are similar to those reported here for the hydrolytic dechlorination of 3-chloroacrylate (⌬H ‡ ϭ 26.7 kcal͞mol; T⌬H ‡ ϭ Ϫ6.5 kcal͞mol). [The possibility that these rates might be similar was predicted by Azurmendi et al (8). ]…”

“…The entirely enthalpic basis of this rate enhancement would seem understandable if polar forces of attraction (rather than substrate approximation or hydrophobic effects) were responsible for binding the altered substrate in the transition state. The active site of CaaD is lined with charged residues that cannot be altered without inactivating the enzyme and are believed to stabilize the transition state in this way (3,4,8). .…”

“…These are not the properties expected of a newly evolved enzyme. Because CaaD lacks any apparent leaving-group site for chloride, and for other reasons, it has been suggested that the apparently novel catalytic activity of CaaD may be a fortuitous side reaction of an enzyme that evolved to act on a natural substrate that remains to be identified (8). That possibility would be strengthened if the gene for CaaD were found to exist in pseudomonads from fields that had never been treated with pesticide.…”

Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase

“…The cis-and trans-3-chloroacrylic acid dehalogenases (cisCaaD and CaaD) 4 catalyze the cofactor-independent hydrolytic dehalogenation of, respectively, the cis-and trans-isomers of 3-chloroacrylic acid (1 and 2, Scheme 1) to produce malonate semialdehyde (5) and HCl (1)(2)(3). Both reactions may be initiated by the attack of water at C3 to form an enzyme-stabilized enediolate intermediate (3).…”

“…Both reactions may be initiated by the attack of water at C3 to form an enzyme-stabilized enediolate intermediate (3). Subsequent ketonization of 3 with protonation at C2 generates a chlorohydrin intermediate (4), which can collapse by direct expulsion of the chloride to afford 5 (1,4,5). Alternatively, ketonization of 3 can result in chloride loss and the formation of the enol intermediate, 6, which tautomerizes to afford 5.…”