Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex

Michael R. Sierks, Birte Svensson

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

Molecular recognition and site-directed mutagenesis are used in combination to identify kinetically, transition state interactions between glucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180 - Gin GA had indicated a role in substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193-198). Here, changes in activation energies calculated from measured kcat/Km values for a series of deoxygenated maltose analogues indicate hydrogen bonding between the mutant enzyme and the 3-OH group of the reducing end sugar ring. Using the same substrate analogues and determining activation energies with wild-type GA an additional hydrogen bond with the 2-OH group of maltose is attributed to an interaction with the carboxylate Glu180. This novel combination of molecular recognition and site-directed mutagenesis enables an enzyme substrate transition state contact to be identified and characterized even without access to the three dimensional structure of the enzyme. Given the distant structural relationships between glucoamylases and several starch hydrolases (Svensson, B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activity of these related enzymes.

Original languageEnglish (US)
Pages (from-to)185-188
Number of pages4
JournalProtein Engineering, Design and Selection
Volume5
Issue number2
DOIs
StatePublished - Mar 1 1992
Externally publishedYes

Keywords

  • Active site mutant
  • Hydrogen-bond energy
  • Kinetics
  • Molecular recognition

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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