Catalytic strategies in schiff-base enzymes

Karen N. Alien, Andrew S. Mazurkie, Marc Morais, Angela S. Baker, Debra Dunawav-Mariano

Research output: Contribution to journalArticle

Abstract

In order to explore the mechanisms by which enzymes can control the formation and subsequent reactivity of Schiff-base intermediates derived from enzymic amines and substrate, a combination of X-ray crystallography and kinetics has been used on three Schiff-base utilizing enzymes: fructose-1,6 -bisphosphate aldolase, phosphonoacetaldehyde hydrolase and acetoacetate decarboxylase. Especially in question is how these enzymes 1) control the nucleophilicity of the active site amine by perturbing the pK of the amino group and 2) prevent the deprotonation of the intermediate imine nitrogen (possibly by shielding the substrate adduct from solvent). Site directed mutagenesis has been used to alter the proposed pK perturbing lysine and the altered activity of the mutants has been measured by examining turnover with substrate as well as by measuring solvent exchange with the adduct formed with product and intermediate/transition state analogues. These experiments also address the relative stability of the substrate and product Schiffbase adducts. The structures of the wild-type enzymes have been determined in the presence of intermediate/transition-state analogues by X-ray crystallography. In the case of mutants with no detectable activity, the substrate has been cocrystallized with the enzyme. The electrostatics of the active site, including solvent accessibility have been calculated for these structures in order to probe the protonation state of the free amine and the imine adduct. We gratefully acknowledge funding by The National Science Foundation MCB 9630430.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Alien, K. N., Mazurkie, A. S., Morais, M., Baker, A. S., & Dunawav-Mariano, D. (1998). Catalytic strategies in schiff-base enzymes. FASEB Journal, 12(8).