:: Mechanism of Enzymatic Catalysis of Glycosyl Transfer: β-Galactosidase.
Many of the details of the mechanism for enzymatic catalysis of glycosyl
transfer reactions are not well understood. Scheme 1 shows a working model
for the first step for catalysis of glycosyl transfer with retention of
configuration, which was first proposed after examination of an X-ray
crystal structure of a lysozyme-inhibitor complex. This proposal is now
supported by extensive results of model studies of nonenzymatic catalysis
of acetal cleavage.
This reaction mechanism is defined by the imperatives
for catalysis of nucleophilic substitution of a poor alkoxide ion leaving
group at glycosides, and these imperatives will probably ensure the
presence of the following essential catalytic residues or metal cofactors
at all enzymes that catalyze glycosyl transfer with retention of
configuration at the glycosidic carbon: (1) A nucleophilic residue that
participates either by providing assistance to expulsion of the leaving
group from the anomeric carbon, and/or electrostatic stabilization of an
oxocarbenium ion reaction intermediate. (2) An acidic residue (e.g., a
carboxylic acid or a metal ion) that provides stabilization of negative
charge at the oxygen leaving group.
In the second step for the hydrolysis reaction, the residue that functions
in catalysis of leaving group expulsion acts as a catalyst of the addition
of water (ROH = HOH) to the glycosyl-enzyme intermediate. Our work on this
enzyme has focused on several problems.
(1) The characterization of the effect of changing alkyl alcohol leaving
group/nucleophile on catalytic activity. See (a)
11703-11712 (1995) and (b)
(2) Site-directed mutagenesis studies that have provided insight into the
mechanism for the enhancement of acid-base catalysis by β-galactosidase.
Biochemistry, 37, 4305-4309 (1998) and (b)
Bioorganic Chemistry, 8, 146-155
(3) The 28000-fold difference in the values of kS = 0.0046/s for
hydrolysis of the 2-deoxy galactosyl-enzyme intermediate of β-galactosidase
and 1300/s for hydrolysis of the intermediate of the physiological
reaction, is consistent with at least a 7.4 kcal/mole stabilization of the
transition state for the physiological reaction by interaction of
β-galactosidase with the C-2 hydroxyl group. This binding interaction will
be even greater than 7.4 kcal/mole, if the inductive effect of the C-2
hydroxyl group causes the same ca. 1000-fold reduction in the rate constant
for sugar hydrolysis as observed for the physiological reaction. By
comparison, it has been estimated that glycosyl transferases provide a ca.
20 kcal/mol stabilization of the transition state for cleavage of simple
glycosides, which corresponds to a 20 kcal/mol transition state binding
energy. In the case of b-galactosidase these binding interactions involve
largely the protein and the β-D-galactopyranosyl group of substrate because
this enzyme is an efficient catalysis of cleavage of alkyl
β-D-galactopyranosides, where the leaving group is a small alkyl alcohol
such as ethanol and trifluoroethanol. The large fraction of this transition
state stabilization that originates in some manner from interaction with a
single C-2 hydroxyl group is striking, but poorly understood and is
currently under investigation in our laboratory.
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