Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity.
Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1071, USA.
Gerrit J
GJ
Poelarends
Hector
H
Serrano
William H
WH
Johnson
Christian P
CP
Whitman
D000480:Alkynes / Q000737:chemistry*; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011422:Propionates / Q000737:chemistry*; D011549:Pseudomonas / Q000201:enzymology
D000480:Alkynes; D011422:Propionates; D002262:Carboxy-Lyases; C071850:malonate semialdehyde decarboxylase; C011537:propiolic acid