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Antibody‐Catalyzed Benzoin Oxidation as a Mechanistic Probe for Nucleophilic Catalysis by an Active Site Lysine
Author(s) -
Sklute Genia,
Oizerowich Rachel,
Shulman Hagit,
Keinan Ehud
Publication year - 2004
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200305034
Subject(s) - chemistry , schiff base , active site , ketone , catalysis , nucleophile , substituent , medicinal chemistry , aldehyde , aldol condensation , stereochemistry , photochemistry , organic chemistry
Aldolase antibody 24H6, which was obtained by reactive immunization against a 1,3‐diketone hapten, is shown to catalyze additional reactions, including H/D exchange and oxidation reactions. Comparison of the H/D exchange reaction at the α‐position of a wide range of aldehydes and ketones by 24H6 and by other aldolase antibodies, such as 38C2, pointed at the significantly larger size of the 24H6 active site. This property allowed for the catalysis of the oxidation of substituted benzoins to benzils by potassium ferricyanide. This reaction was used as a mechanistic probe to learn about the initial steps of the 24H6‐catalyzed aldol condensation reaction. The Hammett correlation ( ρ =4.7) of log( k cat ) versus the substituent constant, σ , revealed that the reaction involves rapid formation of a Schiff base intermediate from the ketone and an active site lysine residue. The rate‐limiting step in this oxidation reaction is the conversion of the Schiff base to an enamine intermediate. In addition, linear correlation ( ρ =3.13) was found between log( K M ) and σ , indicating that electronic rather than steric factors are dominant in the antibody–substrate binding phenomenon and confirming that the reversible formation of a Schiff base intermediate comprises part of the substrate‐binding mechanism.