Biomedical Engineering Reference
In-Depth Information
transition.state.(Benkovic.and.Hammes-Schiffer.2003;.Garcia-Viloca.et.al..2004)..Therefore,.
molecules.that.speciically.bind.and.stabilize.a.transition.state.analog.(TSA).of.a.desired.
reaction.are.good.candidates.to.act.as.enzymes.for.that.reaction..This.strategy.forms.the.basis.
for.catalytic.antibodies:.Designing.and.synthesizing.a.stable.TSA.of.the.desired.reaction.
and.then.using.this.molecule.as.an.antigen.in.immunizations.of.animals.raises.antibodies.
capable.of.stabilizing.the.transition.state.of.the.desired.reaction..Between.approximately.
10
8
.and.10
11
.different.speciicities.are.present.in.a.human.antibody.repertoire.(Hanson.et.al..
2005)..These.varied.libraries.should.contain.a.catalytic.antibody.for.virtually.any.chemical.
reaction.. Catalytic. antibody. generation. is. a. knowledge-driven. method. (Golynskiy. and.
Seelig.2010),.since.construction.of.a.“good”.TSA.requires.a.detailed.understanding.of.the.
reaction.mechanism.
The. irst. catalytic. antibodies,. or. “abzymes,”. were. generated. 25. years. ago. to. catalyze.
the. hydrolysis. of. esters. and. carbonates.. TSAs. were. used. as. haptens. to. produce. mono-
clonal.antibodies.with.the.ability.to.enhance.the.rate.of.the.reactions.∼10
3
-fold.(Pollack.
et.al..1986;.Tramontano.et.al..1986)..Since.then,.artiicial.antibodies.have.been.generated.
that. catalyze. a. plethora. of. chemical. transformations,. including. hydrolysis. of. amides.
and. esters,. cyclization,. decarboxylation,. lactonization,. peroxidation,. and. reactions. for.
which. no. natural. or. artiicial. enzyme. exists. (Nevinsky. et. al.. 2002).. However,. even. the.
most. tailored. abzymes. cannot. outperform. highly. evolved. natural. enzymes.. Abzymes.
achieve.maximum.rate.enhancements.of.2.3.×.10
8
.s
−1
.versus.7.×.10
19
.s
−1
.for.natural.enzymes.
(Golynskiy.and.Seelig.2010).
There. are. several. drawbacks. in. the. design. of. abzymes. that. could. explain. their. lower.
catalytic. eficiency.. Abzymes. are. speciically. designed. to. bind. to. the. TSA,. which. could.
result.in.enzymes.that.bind.too.tightly.to.the.transition.state,.blocking.catalysis.or.product.
release.(Golynskiy.and.Seelig.2010)..Also,.transition.state.stabilization.is.only.one.of.many.
strategies.natural.enzymes.use.to.accelerate.reactions..Abzymes.are.restricted.to.the.single.
immunoglobulin.fold.(Golynskiy.and.Seelig.2010),.and.therefore.have.low.lexibility.and.
plasticity.(Belogurov.et.al..2009).and.solvent-exposed.active.sites.(Xu.et.al..2004)..Abzymes.
have.an.advantage,.however,.as.in.vivo.therapeutic.agents,.since.antibodies.are.less.likely.
than. other. artiicial. enzymes. to. elicit. an. immune. response. in. the. body. (Golynskiy. and.
Seelig.2010).
3.6 De Novo Design of Metalloenzymes
If. protein. active. sites. were. constrained. to. use. only. the. chemical. groups. offered. by. the.
amino.acids,.they.would.not.be.able.to.catalyze.all.the.reactions.required.to.sustain.life.
(Bertini. et. al.. 2007).. As. much. as. 30%. of. all. natural. enzymes. are. thought. to. incorporate.
metal.cofactors.(Ragsdale.2006)..The.chemical.diversity.of.the.inorganic.elements.allows.
these. so-called. metalloenzymes. to. perform. a. wider. range. of. biochemical. functions. by.
facilitating. reactions. such. as. bond. forming. and. breaking,. electron. transfer,. and. radical.
chemistry.(Ragsdale.2006).
The. design. of. artiicial. metalloenzymes. is. challenging.. In. addition. to. providing. a.
stable.protein.scaffold,.the.design.must.place.the.metal.cofactor.in.the.correct.geometry.
and. within. the. proper. environment. to. obtain. the. desired. function.. As. opposed. to.
traditional. metal. catalysts. in. which. the. irst. coordinating. shell. (the. chelating. ligands).
is. the. major. component. that. deines. the. catalytic. activity. and. speciicity,. in. enzymatic.
