Handbook of Solvents - George Wypych - ChemTech - Ventech!

14.4.3 Choice of solvent for enzymatic reaction 877
2) Specificity and selectivity, k cat/K m
It is the most exciting and significant feature that the substrate specificity, enantioselectivity
and regioselectivity can be profoundly affected by nature of solvents in which the enzyme
molecule exists. This phenomenon has opened an alternative approach for changing specificity
and selectivity of an enzyme other than both screening from nature and protein engineering
in the field of synthetic organic chemistry. The ability of enzymes to discriminate
substrate specificity among different, but
structurally similar substrates,
enantioselectivity among enantiomers,
eantiofaces or identical functional groups
linked to a prochiral center, and
regioselectivity among identical functional
groups on the same molecule, is expressed
quantitatively on E value, which is the ratio
of the specificity constants, k cat/K m, for the
two kinds of substrate (or entiomers), i.e.,
(k cat/K m) 1/(k cat/K m) 2. For kinetic resolution
of racemic mixture by the enzyme, E is
called enantiomeric ratio. The higher E, the
higher the enantiomeric excess (i.e., the
optical purity), ee, of the product (or remaining
substrate). It is said that an E value
higher than 100 is preferable for pharmaceutical
or biotechnological applications.
For overview of this topic, see Refs. 13 and
14.
(2a) Substrate specificity
Zaks and Klibanov reported that the substrate
specificity of α-chymotrypsin,
subtilisin, and esterase changed with an organic
solvent. 15 The substrate specificity in
octane was reversed compared to that in
Figure 14.4.3.2. The dependence of (A) subtilisin
Carlsberg and (B) a-chymotrypsin substrate specificity
for substrates 1 and 2 on the ratio of their Raoult’s law activity
coefficients. For the structures of the substrates 1
and 2, and the solvents a through m in (A) and a to g in
(B), refer to Ref. 16. [Adapted, by permission, from C.R.
Wescott and A.M. Klibanov, Biotechnol. Bioeng., 56,
343(1997)].
water. A thermodynamical model that predicted
the substrate specificity of subtilisin
Carlsberg and α-chymotrypsin in organic
media on the basis of specificity of the enzyme
in water and physicochemical characteristics
of the solvents was developed
by Wescott and Klibanov. 16 They determined
k cat/K m for the transesterification of
N-acetyl-L-phenylalanine and N-acetyl-L-serine with propanol in 20 anhydrous solvents,
and correlated the data of (k cat/K m) Ser/(k cat/K m) Phe, first with the solvent to water partition coefficients
for the substrate, P Phe/P Ser. Later they examined the selectivity of subtilisin toward
two different substrates with the Raoult’s law activity coefficients, γ, by the following equation:
15