Mechanism-based catalytic antibody inactivation

Area of Science:

• Biochemistry
• Immunology
• Enzymology

Background:

• Catalytic monoclonal antibodies (isoabzymes) are crucial tools for understanding biochemical reactions.
• Ester hydrolysis is a fundamental enzymatic process with implications in various biological systems.

Purpose of the Study:

• To elucidate the mechanism of ester hydrolysis by catalytic monoclonal antibodies 18H4, 7D4, and 45A11.
• To investigate the interaction of alternative substrates with these isoabzymes.

Main Methods:

• Utilized alternative substrates, N-acetylglycine phenyl ester (3) and N-carbobenzoxy-glycine phenyl ester (4), to study antibody activity.
• Performed kinetic analyses to determine catalytic constants and inactivation parameters.
• Investigated antibody inactivation and acylation through turnover studies.

Main Results:

• All three antibodies effectively catalyzed the hydrolysis of substrate 3, showing kinetics similar to phenyl acetate.
• Substrate 4 acted as a mechanism-based inactivator for antibody 18H4, with specific kinetic constants (kinact = 0.29 min-1, K' = 64 microM).
• Antibody 18H4 inactivation by substrate 4 involved acylation of tyrosines within the combining site after approximately 3.6 turnovers.

Conclusions:

• The study provides a detailed understanding of ester hydrolysis mechanisms employed by catalytic antibodies.
• The inactivation of antibody 18H4 by substrate 4 suggests a specific reaction pathway involving tyrosyl acylation.
• Findings contribute to the knowledge of antibody-substrate interactions and catalytic mechanisms.