Enzyme Technology
Artificial enzymes
A number of possibilities now exist for the construction of artificial
enzymes. These are generally synthetic polymers or oligomers with enzyme-like
activities, often called synzymes. They must possess two structural entities, a
substrate-binding site and a catalytically effective site. It has been found
that producing the facility for substrate binding is relatively straightforward
but catalytic sites are somewhat more difficult. Both sites may be designed
separately but it appears that, if the synzyme has a binding site for the
reaction transition state, this often achieves both functions. Synzymes
generally obey the saturation Michaelis-Menten kinetics as outlined in Chapter
1. For a one-substrate reaction the reaction sequence is given by
synzyme + S
(synzyme-S complex)
synzyme + P [8.5]
Some synzymes are simply derivatised proteins, although covalently
immobilised enzymes are not considered here. An example is the derivatisation of
myoglobin, the oxygen carrier in muscle, by attaching (Ru(NH3)5)3+ to three
surface histidine residues. This converts it from an oxygen carrier to an
oxidase, oxidising ascorbic acid while reducing molecular oxygen. The synzyme
is almost as effective as natural ascorbate oxidases.
It is impossible to design protein synzymes from scratch with any probability
of success, as their conformations are not presently predictable from their
primary structure. Such proteins will also show the drawbacks of natural
enzymes, being sensitive to denaturation, oxidation and hydrolysis. For example,
polylysine binds anionic dyes but only 10% as strongly as the natural binding
protein, serum albumin, in spite of the many charges and apolar side-chains.
Polyglutamic acid, however, shows synzymic properties. It acts as an esterase in
much the same fashion as the acid proteases, showing a bell-shaped pH-activity
relationship, with optimum activity at about pH 5.3, and Michaelis-Menten
kinetics with a Km of 2 mm and Vmax of 10−4 to
10−5 s−1 for the hydrolysis of
4-nitrophenyl acetate. Cyclodextrins (Schardinger dextrins) are naturally
occurring toroidal molecules consisting of six, seven, eight, nine or ten a-1,
4-linked D-glucose units joined head-to-tail in a ring (a-, b-,
g-, d- and
e-cyclodextrins,
respectively: they may be synthesised from starch by the cyclomaltodextrin
glucanotransferase (EC 2.4.1.19) from Bacillus macerans). They differ in the
diameter of their cavities (about 0.5-1 nm) but all are about 0.7 nm deep. These
form hydrophobic pockets due to the glycosidic oxygen atoms and inwards-facing
C-H groups. All the C-6 hydroxyl groups project to one end and all the C-2 and C-3
hydroxyl groups to the other. Their overall characteristic is hydrophilic, being
water soluble, but the presence of their hydrophobic pocket enables them to bind
hydrophobic molecules of the appropriate size. Synzymic cyclodextrins are usually
derivatised in order to introduce catalytically relevant groups. Many such
derivatives have been examined. For example, a C-6 hydroxyl group of b-cyclodextrin was covalently derivatised by an activated pyridoxal coenzyme.
The resulting synzyme not only acted a transaminase (see reaction scheme [1.2])
but also showed stereoselectivity for the L-amino acids. It was not as active as
natural transaminases, however.
Polyethyleneimine is formed by polymerising ethyleneimine to give a highly
branched hydrophilic three-dimensional matrix. About 25% of the resultant amines
are primary, 50% secondary and 25% tertiary:
[8.6]
Ethyleneimine
polyethyleneimine
The primary amines may be alkylated to form a number of derivatives. If 40%
of them are alkylated with 1-iodododecane to give hydrophobic binding sites and
the remainder alkylated with 4(5)-chloromethylimidazole to give general
acid-base catalytic sites, the resultant synzyme has 27% of the activity of a-chymotrypsin
against 4-nitrophenyl esters. As might be expected from its apparently random
structure, it has very low esterase specificity. Other synzymes may be created
in a similar manner.
Antibodies to transition state analogues of the required reaction may act as
synzymes. For example, phosphonate esters of general formula (R-PO2-OR')− are
stable analogues of the transition state occurring in carboxylic ester
hydrolysis. Monoclonal antibodies raised to immunising protein conjugates
covalently attached to these phosphonate esters act as esterases. The
specificities of these catalytic antibodies (also called abzymes) depends on the
structure of the side-chains (i.e., R and R' in (R-PO2-OR')−) of the antigens.
The Km values may be quite low, often in the micromolar region, whereas the Vmax
values are low (below 1 s−1), although still 1000-fold higher than hydrolysis by
background hydroxyl ions. A similar strategy may be used to produce synzymes by
molecular 'imprinting' of polymers, using the presence of transition state
analogues to shape polymerising resins or inactive non-enzymic protein during
heat denaturation.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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