Reactions of 4-oxalocrotonate tautomerase and YwhB with 3-halopropiolates: analysis and implications.
4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate. The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction. These residues are also critical for the trans-3-chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde. Recently, 3-bromo- and 3-chloropropiolate, the acetylene analogues of 3-haloacrylates, were characterized as potent irreversible inhibitors of CaaD due to the covalent modification of the catalytic proline. In view of these observations, an investigation of the behavior of 4-OT and YwhB with the 3-halopropiolates was undertaken. The results show that these compounds are potent irreversible inhibitors of 4-OT and YwhB with Pro-1 being the sole site of covalent modification by 3-bromopropiolate. The inactivation process could involve the enzyme-catalyzed addition of water to the 3-halopropiolate yielding an acyl halide, which would inactivate the enzyme or be initiated by the nucleophilic attack of Pro-1 at the C-3 position of the 3-halopropiolate in a Michael type reaction. The presence of the halogen along with Arg-11 could facilitate both reactions with the latter causing the polarization of the alpha,beta-unsaturated acids. The 3-halopropiolates are the first identified inhibitors of YwhB and confirm the importance of Pro-1 in its mechanism. In addition, the results set the stage for the use of these compounds as mechanistic probes of the primary as well as low level activities of 4-OT and YwhB.
Wang
Susan C
SC
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Johnson
William H
WH
Czerwinski
Robert M
RM
Whitman
Christian P
CP
D000480:Alkynes / Q000737:chemistry*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D046915:Penicillin-Binding Proteins; D033903:Periplasmic Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000235:genetics; D011422:Propionates / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000480:Alkynes; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C436969:PbpG protein, Bacillus subtilis; D046915:Penicillin-Binding Proteins; D033903:Periplasmic Proteins; D011422:Propionates; D011392:Proline; D006867:Hydrolases; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C077719:cis-3-chloroacrylic acid dehalogenase; C046137:haloalkane dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; C011537:propiolic acid