TY - JOUR
T1 - Presteady-state kinetics of Bacillus 1,3-1,4-β-glucanase
T2 - Binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate
AU - Abel, M.
AU - Planas, A.
AU - Christensen, U.
PY - 2001/7/1
Y1 - 2001/7/1
N2 - In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s-1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.
AB - In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s-1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.
KW - Fluorescence
KW - Induced-fit
KW - Inhibition
KW - Mechanism
KW - Stopped-flow
UR - http://www.scopus.com/inward/record.url?scp=0035395783&partnerID=8YFLogxK
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=pure_univeritat_ramon_llull&SrcAuth=WosAPI&KeyUT=WOS:000169783500021&DestLinkType=FullRecord&DestApp=WOS_CPL
U2 - 10.1042/0264-6021:3570195
DO - 10.1042/0264-6021:3570195
M3 - Article
C2 - 11415449
AN - SCOPUS:0035395783
SN - 0264-6021
VL - 357
SP - 195
EP - 202
JO - Biochemical Journal
JF - Biochemical Journal
IS - 1
ER -