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

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• 作者：Hugo F. Azurmendi ; Susan C. Wang ; Michael A. Massiah ; Gerrit J. Poelarends ; Christian P. Whitman ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：April 13, 2004
• 年：2004
• 卷：43
• 期：14
• 页码：4082 - 4091
• 全文大小：203K
• 年卷期：v.43,no.14(April 13, 2004)
• ISSN：1520-4995

文摘

trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonatesemialdehyde by the addition of H₂O to the C-2, C-3 double bond, followed by the loss of HCl from theC-3 position. Sequence similarity between CaaD, an (s/gifchars/alpha.gif" BORDER=0>s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">)₃ heterohexamer (molecular weight 47 547),and 4-oxalocrotonate tautomerase (4-OT), an (s/gifchars/alpha.gif" BORDER=0>)₆ homohexamer, distinguishes CaaD from those hydrolyticdehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaDdemonstrates that s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">Pro-1 (i.e., Pro-1 of the s/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> subunit), s/gifchars/alpha.gif" BORDER=0>Arg-8, s/gifchars/alpha.gif" BORDER=0>Arg-11, and s/gifchars/alpha.gif" BORDER=0>Glu-52 are at or near theactive site, and the s/entities/ge.gif">10^3.4-fold decreases in k_cat on mutating these residues implicate them as mechanisticallyimportant. 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_aof 9.2. NMR titration of ¹⁵N-labeled wild-type CaaD yielded pK_a values of 9.3 and 11.1 for the N-terminalprolines, while the fully active but unstable s/gifchars/alpha.gif" BORDER=0>P1A mutant showed a pK_a of 9.7 (for the s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">Pro-1), implicatings/gifchars/beta2.gif" BORDER=0 ALIGN="middle">Pro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the firstevidence 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 forthe general base in CaaD is the active site residue s/gifchars/alpha.gif" BORDER=0>Glu-52. CaaD has 10 arginine residues, six in thes/gifchars/alpha.gif" BORDER=0>-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). ¹H-¹⁵N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showedseven to nine Arg-Ns/gifchars/epsilon.gif" BORDER=0 >H 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 s/gifchars/alpha.gif" BORDER=0>R11A mutant (s/gifchars/delta.gif" BORDER=0 >H = 7.11ppm, s/gifchars/delta.gif" BORDER=0 >N = 89.5 ppm), and another one (R_G) disappeared in the spectrum of the inactive s/gifchars/alpha.gif" BORDER=0>R8A mutant(s/gifchars/delta.gif" BORDER=0 >H = 7.48 ppm, s/gifchars/delta.gif" BORDER=0 >N = 89.6 ppm), thereby assigning these resonances to s/gifchars/alpha.gif" BORDER=0>Arg-11Ns/gifchars/epsilon.gif" BORDER=0 >H, and s/gifchars/alpha.gif" BORDER=0>Arg-8Ns/gifchars/epsilon.gif" BORDER=0 >H, respectively. ¹H-¹⁵N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoicacid (3-CBA), a competitive inhibitor (subscribe/journals/bichaw/43/i14/eqn/bi030241ue10001.gif"> = 0.35 ± 0.06 mM), resulted in progressive downfieldshifts of the s/gifchars/alpha.gif" BORDER=0>Arg-8Ns/gifchars/epsilon.gif" BORDER=0 > resonance yielding a K_D = 0.77 ± 0.44 mM, comparable to the subscribe/journals/bichaw/43/i14/eqn/bi030241ue10002.gif">, suggestiveof active site binding. Increasing the pH of free CaaD to 8.9 at 5 s/entities/deg.gif">C resulted in the disappearance of allnine Arg-Ns/gifchars/epsilon.gif" BORDER=0 >H resonances due to base-catalyzed Ns/gifchars/epsilon.gif" BORDER=0 >H exchange. Saturating the enzyme with 3-CBA (16mM) induced the reappearance of two Ns/gifchars/epsilon.gif" BORDER=0 >H signals, those of s/gifchars/alpha.gif" BORDER=0>Arg-8 and s/gifchars/alpha.gif" BORDER=0>Arg-11, indicating that thebinding of the substrate analogue 3-CBA selectively slows the Ns/gifchars/epsilon.gif" BORDER=0 >H exchange rates of these two arginineresidues. The kinetic and NMR data thus indicate that s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">Pro-1 is the acid catalyst, s/gifchars/alpha.gif" BORDER=0>Glu-52 is a reasonablecandidate for the general base, and s/gifchars/alpha.gif" BORDER=0>Arg-8 and s/gifchars/alpha.gif" BORDER=0>Arg-11 participate in substrate binding and in stabilizingthe aci-carboxylate intermediate in a Michael addition mechanism.