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Enzyme Mimics.

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Enzyme Mimics
Anthony J. Kirby*
One of the great intellectual challenges presented to Science
by Nature is a proper understanding of how enzymes work. At
one level we can “explain” enzyme catalysis -what an enzyme
does is bind. and thus stabilize selectively, the transition state for
a particular reaction.“] But our current level of understanding
fails the more severe, practical test--that of designing and making artificial enzyme systems with catalytic efficiencies which
rival those of natural enzymes.
Enzyme mimics have long been high-profile targets for bioorganic chemists. The picture to date has been the familiar one
of steady progress, with occasional flashes of inspiration, and a
heightening awareness of just how complex the problem is. But
two recent reports”, 31-approaching the problem from completely different directions- claim catalytic efficiencies in artificial systems which are extraordinary in terms of conventional
wisdom. First we should set the scene.
The ”conventional wisdom” is based on the one hand on
mechanistic work on enzymes, and on the other hand on (mostly
separate) studies of binding and catalysis in simpler, artificial
systems. Enzymes are of course more than just highly evolved
catalysts : they also recognize and respond to molecules other
than their specific substrate and product, as part of the control
mechanisms of the cell. But the evolution of enzyme mimics is
at a stage where the efficient combination of binding and catalysis is the main objective. A starting point may be a system
which shows efficient binding; or the mechanism of the reaction
concerned, since it is the rate-determining transition state which
is the most important target for the binding process. Eventually
the two approaches must converge if a genuine enzyme mimic is
to emerge. This sort of work holds out the promise of artificial
catalysts. which may be more robust than proteins, for unnatural reactions of interest. On the other hand, the most natural
basis for an enzyme mimic is inevitably a real enzyme. All these
approaches are currently producing interesting results.
Enzyme-Based Mimics
It is possible to modify a natural enzyme, chemically or more
commonly by the methods of protein engineering, in such a way
that its specificity is altered, even to the point that the modified
system will catalyze a new reaction. Such systems are modified
enzymes rather than enzyme mimics, which to qualify in this
context should have been constructed artificially.
When it became possible to identify the functional groups in
enzyme active sites it was natural to look at small peptides
containing active-site sequences of amino acids as possible catalysts. The results of this sort of work were uniformly negative.
We know now that a working enzyme active site has its functional groups disposed in a specific, dynamic three-dimensional array, with significant interactions with the rest of the protein; this
cannot successfully be inodeled in two dimensions.
Dr. A . 1. Kirby
Universi t!, Chemlcal Laboratory
GB-Carnbridpe CB2 1EW (UK)
Telefax I n t code f ( 2 2 3 ) 336362
A true enzyme-based mimic might try to reproduce the threedimensional arrangement of the functional groups of the active
site in a synthetic framework. The reasoning is simple; turning
the idea into real molecules less so. However, what appears to be
a major success for this approach is the report of Atassi and
Manshouri’’] on the preparation of two so-called “pepzymes”,
modeled from the active site structures of trypsin and chymotrypsin by “surface-simulatioii”. This involved the design
and synthesis of a series of relatively small (29 residue) peptides
containing the key catalytic and binding amino acid sequences
of the enzymes. These were connected by glycine spacers so as
to model the three-dimensional arrangement known from the
X-ray structures of the enzyme and its complexes with substrate
analogues. An early version which showed some trypsin-like
binding activity was modified systematically to the point where
the peptide (native active site residues indicated in bold) shows
extraordinary catalytic activity, specifically in the cyclic (disulfide) form A.
- Gly-
Not only does this molecule hydrolyze the simple trypsin
“substrate” N-tosyl-L-arginine methyl ester with k,,, and K,,,
comparable to those of the native enzyme, but it also hydrolyzes
test proteins to give similar peptide profiles. A peptide closely
related to A based on the chymotrypsin active site had similar
activity and the expected, different specificity.
I t must be said that this level of activity is surprising, especially against amide bonds, and intensive efforts to repeat the results are under way. If confirmed this work will be seen as an
important advance: the design stage may be complex, but with
modern synthetic methods peptides of this sort of size are quite
reasonable synthetic targets. A practical limitation, as always
with enzyme-based mimics, is that--at this stage of development at least--complete success simply means doing a reaction
as well as an available natural catalyst.
Mechanism-Based Mimics
At the other extreme, it is possible to achieve enormous rate
accelerations in quite simple systems by by-passing the binding
process and making reactions intramolecular, that is, by bringing the functional groups concerned together on the same mole ~ u l e . Typically,
making the reaction of interest part of a
thermodynamically favorable cyclization can produce systems
in which the extraordinarily stable groups of structural biology
(amides, glycosides, and phosphate esters have half-lives of
many years under physiological conditions near pH 7) can be
cleaved in a fraction of a second. Detailed chemical mechanisms
of catalysis can then be worked out for specific reactions, studied under the same conditions and proceeding at similar rates
as the same reactions between the same two (or more) functional
groups in enzyme active sites. Two measures of catalytic efficiency are relevant: the effective molarity (EM), that is the effective
concentration of the catalytic group. that would be needed to
make an intermolecular reaction go at the rate of the intramolecular
and, of course, the absolute rate of the
reaction. Because reactions in enzyme active sites are very fast:
fast enough for many enzymes to have reached evolutionary
perfection, defined by Albery and Knowlesrsl as catalytic efficiency so high that the rate-determining step of the reaction
concerned is diffusion away of the products.
E M values as high as 3 0' - l0l4 M-meaning half-lives of the
order of a second--can be attained in systems where on ordinary aliphatic amide is forced into close proximity with a
or a phosphate diester unit with a neighboring OH,['] and it is possible to define detailed mechanisms for
such model reactions. These serve as an essential basis for the
discussion of the mechanisms of the same reactions in enzyme
active sites, or for the design of enzyme mimics. It is not possible- so far at least-to attain rate-enhancements of anything
like this magnitude when the reacting groups are brought together by noncovalent binding.
Binding-Step Based Mimics
A minimum requirement for a true enzyme mimic is a binding
interaction between two molecules prior to the catalytic reaction, indicated by Michaelis-Menten kinetics. Intramolecular
systems can support very rapid reactions because we can use
synthesis to bring groups together into close and unavoidable
proximity. But an enzyme must select and bind its substrate
noncovalently in a dynamic equilibrium. The chemistry of the
molecular recognition processes involved is one of the most
active areas of current research, and a popular topic for meetings,i8. '] highlights,['01and reviews.[' '] As with chemical catalysis, much of our understanding of noncovalent interactions
comes from studies of simple systems designed to answer
specific questions about the basic processes. More directly relevant to the development of enzyme mimics are systems designed
to achieve catalysis by binding. that is without specific catalytic
groups built in. These fall into two important classes: synthetic
hosts designed to bring two reactants into close and productive
proximity, and most catalytic antibodies.
The majority of catalytic antibodies['2. l3]so far known have
been designed to catalyze the hydrolysis of carboxylic acid
derivatives, and have been raised against phosphonate haptens
2. which model the structure of the tetrahedral transition states
involved ( I ) . Catalysis of ester hydrolysis is rather reliably obtained with suitable haptens (and substrates), with the nucleophile coming from the solvent. More ambitious systems with
catalytic groups built-in can. in principle, be obtained by careful
hapten design (coupled with a large slice of luck); or from an
existing catalytic antibody by protein engineering. This is an
area of definite promise, and much current activity.
Careful hapten design also allowed the preparation of an
antibody that catalyzed the Diels- Alder reaction of tetrachlorothiophene dioxide 3 and N-ethylmaleimide.['4~ Again,
catalysis results simply from productive binding (an EM of
> 110 M is estimated): turnover depends on the instability of the
initial adduct 4, which loses SO, very rapidly to give the product
5. This avoids product inhibition, which is a common problem with such potential catalytic systems: the hapten 6
(R = (CH,),CO,H) is a reasonable transition state analogue,
but geometrically very different from the final product.
The Diels- Alder reaction can also be catalyzed by simple
artificial systems. A recent example is the reversible reaction
between 7 and 8, which is accelerated by (stoichiometric
amounts of) a cyclic zinc-porphyrin trimer host which binds
pyridine derivatives inside the cavity.['61 The product is the exo
adduct, produced up to 1000 times faster than the corresponding endo isomer (which is obtained as the kinetic product in the
absence of the macrocycle). This corresponds to an EM of
about 20 M. The system is not catalytic because of product inhibition (as was an early example using the cavity of a cyclodextrin
as host["').
Enzyme Mimics Showing Binding and Catalysis
Many of the most successful enzyme mimics have involved
functionalized cyclodextrins, and the work of Breslow et al.. in
B 10.00
+ .?5,'11
Angaw Chem. Inl. Ed. Engl. 1994, 33. No. S
particular, is familiar to anyone who has followed the field.“
These hosts bind aromatic rings within a hydrophobic cavity. In
another seminal contribution Lehn et al.[19,201 used polyammonium macrocycles to catalyze phosphate transfer reactions of ATP, demonstrating that multiple hydrogen bonds can
also be an effective source of binding between flexible systems in
aqueous solution.
A different approach has been reported by Benner et al.,[’’]
who based the design of a synthetic decarboxylase on the known
properties of proteins and the mechanism of amine-catalyzed
decarboxylation of 8-ketoacids. Their enzyme mimics are 14residue peptides (called oxaldie 1 and 2) based on leucine and
lysine. in a sequence known to favor x-helix formation, and thus
expected to adopt protein-like conformations, with a hydrophobic core and a hydrophilic exterior. They catalyze the decarboxylation of oxalylacetate by the expected mechanism (see
Scheme 1). with the cationic lysine side chains presumably involved in binding the two carboxylates of both substrate and
transition state (acetoacetate. with a single carboxylate group, is
not a substrate). Michaelis-Menten kinetics are observed and
Scheme 1 . Dccarhoxylation of oxalylacetate cdtdlyzed by an enzyme mimic.
R = lysine 5ide chain.
more effective than acetate. Interestingly the reaction is stoichiometric : though a mixed anhydride is almost certainly an
intermediate, its hydrolysis must be rate-determining in the
overall hydrolysis of the anilide. The authors suggest that the
reaction takes place in submicellar aggregates or “clumps”, in
which hydrophobic association of the long alkyl chains brings
anilide carbonyl and hexadecanoate carboxylate groups into
close and remarkably productive proximity (see Scheme 2).
(Simply adding three methylene groups to the substrate [9,
X = (CH,),NH] eliminates the observed reaction.)
Scheme 2. Hydrophobic association between 9 and hexadecanoate
Two points are of special interest here: the high reactivity,
which is much greater than previously observed for such apparently loosely-associated systems : and the principle of screening
large numbers of simple systems, rather than actually synthesizing complex, carefully designed ones. The newer approach supplements existing ways of thinking, and practical applications
may result.
German version: Angekr. Chem. 1994. 106. 573
imine formation is 103-104 times faster than with simple amine
catalysts. Also the activity does indeed seem to depend on the
degree of 9-helix formation.
The simplest. and one of the most remarkable, new enzyme
mimics has emerged from a piece of “lateral thinking” by
Menger and Fei.[3]No synthesis is involved. These authors simply mixed long-chain carboxylic acids, amines, alcohols, and
alkyliniidazoles. of the sort known to form aggregates, and
eventually micelles, in aqueous solution : then screened large
numbers of such mixtures for catalytic activity. The test reaction
was the hydrolysis of the reactive ester 9 (X = O ) ,which is easily
followed above pH 7 by the release of the p-nitrophenolate
chromophore. Some of the mixtures used effected the hydrolysis
of 9 (X = 0) at rates too fast to measure manually. Remarkably
this was also true in the
presence of a single compo, N ~l ~ , C , Ho , N O ~
nent when this was the hex-
adecanoate anion. and this
system also effects the hydrolysis of the p-nitroanilide (9, X = NH). This is an activated
amide, but one that is not hydrolyzed detectably in the presence
of 0.2 M acetate at p H 7 (25 “C). But under similar conditions, in
the presence ofjust 2 x
M hexadecanoate its half-life is only
three minutes.
Only nucleophilic catalysis can account for such an efficient
p r o ~ e s s . ’N~ o] EM can be calculated as no data are available for
a suitable comparison, but hexadecanoate is at least 1O8 times
[ l ] A. R. Fersht. Enzyme Structrrre mil Mechunism. second edition, W. H . Freeman, New York, 1985.
[2] M. Z. Atassi. T. Manshouri, Proc. Null. Acud. Sci. U S A , 1993, YO, 8282 -8286.
[3] F. Menger, 2. X. Fei, Angew. Chem. 1994. 106. 329; Angeiv. Clirm. 1111.Ed.
Engl. 1994, 33, 346.
[4] A. J. Kirby, Effective molurities for inlruniolecular reactions (Adv. Phys. Orx.
Chem. 1980, 17, 183-278).
[5] W. J. Albery, J. R. Knowles, Biochemistry 1976, 15, 5631
[6] F. M. Menger, M. Ladika, J. A m . Chem. Sor. 1988, ffO,6794
[?I K. N. Dalby. A. J. Kirby, F. Hollfelder, J. Chem. SOC.Perkin Guns. 2 1993.
[8] Host-Guest In/eruction.s.-,fromChemistry /o Biolog? (Eds. : D. J. Chadwick, K .
Widdows) (Crhu Found. Svmp. 1991, 158).
[9] Tlic Chemrstrj~of Biologirul Molerulur Recognition (Eds: A. J. Kirby. D. H.
Williams) (Philos. Trans. R. Soc. London A 1993, 345, 1 - 164)
[lo] H.-J. Schneider, Angen. Chem. 1993, 105. 890; Angew. Chfm. I n / . Ed. Engl.
1993, 32, 848.
I l l ] R. J. Pieters, J. Rehek, Red. Trov. Clirm. Puys-Bu.\ 1993, 112. 330; I . Chao. F.
Diederich, ibid. 1993, 112, 335.
1121 P. G . Schultz, Angew. Chem. 1989, 101, 1336; Angew. Chem. Inr. Ed. Engl.
1989,28, 1283.
1131 U. K. Pandit. Recl. Truv. Chim. P a w B u s 1993, f12, 431.
[I41 D. Hilvert, K.W. Hill, K . D. Narel, M.-T. M. Auditor. J. Am. Chern.Soc. 1989.
1f 1. 9261 : see also [I 51.
1151 A. C. Braisted. P. G. Schultz. 1 A m Chem. Suc. 1992, f12% 7431
1161 C. J. Walter. H . L. Anderson, J. K. M. Sanders. 1 Cltern. Soc. Chhem. Commun.
1993, 458, and references therein.
(171 D. Rideout, R. Breslow, J. Am. Chem. Soc. 1980, 102, 7816.
1181 For recent references and a review see: R. Breslow, P. J. Duggan. J. P. Light. J.
Am. Chem. Soc. 1992, 114. 3982; R. Breslow in [8], p. 115.
[19] M. W. Hosseini. J.-M. Lehn. K . C . Jones, K. E. Plute, K. B. Mertes, M. P.
Mertes, 1 Am. Chem. Soc. 1989. ffl. 6330-6335; M. P. Mertes, K.B. Mertes.
Acc. Chrm Res. 1990. 23, 413-418.
[20] K. J o h n s o n , R. K. Allemann, H. Widmer, S. Benner, Nururc, fLondonJ 1993.
365, 530-532.
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