Kinetic and mechanistic characterization of an efficient hydrolytic antibody: Evidence for the formation of an acyl intermediate

Jincan Guo, Wei Huang, Thomas (Tom) Scanlan

Research output: Contribution to journalArticle

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Abstract

In this paper we report the generation and mechanistic characterization of an unusually active catalytic antibody raised to a phosphonate transition-state analog of norleucine. The antibody (17E8) catalyzes the hydrolysis of both norleucine and methionine phenyl esters and is specific for enantiomers that possess the natural S configuration (L) at the α-carbon. The antibody shows side-chain selectivity (kcat/KM) for norleucine over methionine phenyl esters and also shows selectivity for amino acid ester substrates bearing a formyl substituent at the α-amino position. The antibody-catalyzed hydrolysis of all the ester substrates exhibits a bell-shaped pH-rate profile that is consistent with a mechanistic scheme featuring two ionizable active site residues that mediate catalysis. The calculated pKa values for these two residues are 8.9 and 10.1, and in the most catalytically active state of the antibody, the pKa, 8.9 residue is deprotonated and the pKa 10.1 residue is protonated. In the presence of hydroxylamine, partitioning between hydrolysis and hydroxaminolysis is observed, indicating that a covalent acyl intermediate is formed in the antibody-catalyzed reaction. The hydroxaminolysis:hydrolysis product ratio increases with increasing hydroxylamine concentration, while the overall reaction velocity (as measured by phenol release) is unaffected by increasing hydroxylamine concentration. These data support a mechanism involving a covalent acyl intermediate where formation of the intermediate is the rate-determining step in the antibody-catalyzed hydrolysis reaction.

Original languageEnglish (US)
Pages (from-to)6062-6069
Number of pages8
JournalJournal of the American Chemical Society
Volume116
Issue number14
StatePublished - Jul 13 1994
Externally publishedYes

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Antibodies
Antibody Formation
Norleucine
Hydrolysis
Hydroxylamine
Kinetics
Esters
Methionine
Bearings (structural)
Catalytic Antibodies
Organophosphonates
Enantiomers
Substrates
Phenol
Catalysis
Phenols
Amino acids
Catalytic Domain
Carbon
Amino Acids

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Kinetic and mechanistic characterization of an efficient hydrolytic antibody : Evidence for the formation of an acyl intermediate. / Guo, Jincan; Huang, Wei; Scanlan, Thomas (Tom).

In: Journal of the American Chemical Society, Vol. 116, No. 14, 13.07.1994, p. 6062-6069.

Research output: Contribution to journalArticle

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