scholarly journals The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides

1973 ◽  
Vol 133 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Michael L. Sinnott ◽  
Ian J. L. Souchard
Keyword(s):  
Steady State ◽  
Isotope Effects ◽  
Ion Pair ◽  
Enzyme Mechanism ◽  
Kinetic Isotope ◽  
Deuterium Substitution ◽  
Steady State Kinetic ◽  
Hydrolysis Of ◽  
State Kinetic ◽  
Step Mechanism

1. Steady-state kinetic parameters for the β-galactosidase-catalysed hydrolysis of 13 aryl β-d-galactopyranosides show no simple dependence on aglycone acidity. 2. α-Deuterium kinetic isotope effects (kH/kD) for seven of these substrates, measured under steady-state conditions with [S]»Km, vary from 1.00 for poor substrates to 1.25 for hydrolysis of the galactosyl-enzyme. 3. Methanolysis of the galactosyl-enzyme in 1.5m-methanol increases KH/kD for degalactosylation, but leaves that for hydrolysis of ‘slow’ substrates unchanged. 4. These data are incompatible with a simple two-step mechanism. A scheme consisting of a conformation change, liberation of a galactopyranosyl cation in an intimate ion-pair, non-productive but preferential collapse of the ion-pair to a covalent species and reaction of the galactosyl enzyme through the ion-paired form is proposed. 5. This scheme is used to rationalize previously puzzling data about the enzyme mechanism.

scholarly journals Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS

2003 ◽  
Vol 374 (2) ◽  
pp. 369-380 ◽  
Author(s):  
Omid HEKMAT ◽  
Ken TOKUYASU ◽  
Stephen G. WITHERS
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Intrinsic Rate ◽  
Degree Of Polymerization ◽  
Colletotrichum Lindemuthianum ◽  
Chitin Deacetylase ◽  
Steady State Kinetic ◽  
Intrinsic Rate Constant ◽  
Hydrolysis Of ◽  
State Kinetic

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed Km and kcat/Km on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (kcat, 7 s−1) is independent of n and represents the intrinsic rate constant (kint) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed kcat/Km values (A−2, −11.0; A−1, −1.5; A0, −7.7; A+1, −12.5 kJ·mol−1). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [ΔΔG(N-acetyl)=3.3, 0, 4.0 and 0 kJ·mol−1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.

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