KINETIC IDENTIFICATION OF A HYDROGEN-BONDING PAIR IN THE GLUCOAMYLASE-MALTOSE TRANSITION-STATE COMPLEX

Citation
Mr. Sierks et B. Svensson, KINETIC IDENTIFICATION OF A HYDROGEN-BONDING PAIR IN THE GLUCOAMYLASE-MALTOSE TRANSITION-STATE COMPLEX, Protein engineering, 5(2), 1992, pp. 185-188
Citations number
24
Language
INGLESE
art.tipo
Article
Journal title
ISSN journal
0269-2139
Volume
5
Issue
2
Year of publication
1992
Pages
185 - 188
Database
ISI
SICI code
0269-2139(1992)5:2<185:KIOAHP>2.0.ZU;2-1
Abstract
Molecular recognition and site-directed mutagenesis are used in combin ation to identify kinetically, transition state interactions between g lucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180 --> Gln GA had indicated a role in substrate binding for Glu180 (Sierks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193 - 198). Here, changes in activation energies calculated from measured k(cat)/K(m) 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 a nalogues and determining activation energies with wild-type GA an addi tional hydrogen bond with the 2-OH group of maltose is attributed to a n interaction with the carboxylate Glu180. This novel combination of m olecular recognition and site-directed mutagenesis enables an enzyme s ubstrate transition state contact to be identified and characterized e ven without access to the three dimensional structure of the enzyme. G iven the distant structural relationships between glucoamylases and se veral starch hydrolases (Svensson, B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activit y of these related enzymes.