Mechanism of Agrobacterium beta-glucosidase: kinetic studies.
The beta-glucosidase from Agrobacterium faecalis (previously Alcaligenes faecalis) has been subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It has a relatively broad specificity for the substrate sugar moiety and exhibits a classical pH dependence for its kinetic parameters with three different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates has allowed construction of a Bronsted plot, the concave-downward shape of which is consistent with the anticipated two-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Bronsted plot (beta 1g = -0.7) indicates a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for five different aryl glucosides are also consistent with this mechanism and further suggest that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD = 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analogue inhibitors are presented which support the concept of sp2-hybridized transition states.
Kempton
J B
JB
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Withers
S G
SG
D001667:Binding, Competitive; D003903:Deuterium; D005960:Glucosides / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012231:Rhizobium / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005960:Glucosides; C070512:2',4'-dinitrophenylglucopyranoside; D003903:Deuterium; D001617:beta-Glucosidase