Chemistry Reference
In-Depth Information
from.the.initial.tetrahedral.coordination..When.the.constraint.on.RC.is.released,.the.
system.evolves.into.the.product.state.(EP,.Figure.1.7,.Scheme.1.2),.where.OH
−
.reori-
ents. H-bonding. to. Asp120. and. bridging. the. Zn1. and. Zn2. ions. as. in. the. ES. state..
The.CEF.substrate.is.now.completely.hydrolyzed,.the.β-lactam.ring.is.open.and.C8.
acquires.planar.sp
2
.hybridization.and.is.inally.detached.from.the.metal.center..The.
estimated.free.energy.of.activation.(Δ
F
).for.the.single-step.mechanism.is.Δ
F
= 18(±2).
kcal. mol
−1
,. which. is. consistent. with. experimental. evidence. that. suggests. a. single-
step.reaction.for.cephalosporin.hydrolysis.and.a.free.energy.of.activation.of.about.
17.kcal.mol
−1
.for.similar.reactions.
In.mono-Zn.BcII,.Zn1.undergoes.a.large.rearrangement.of.its.coordination.shell.
upon.nucleophilic.attack..When.the.hydroxyl.nucleophile.attacks.the.β-lactam.car-
bonyl. carbon,. Zn1. distorts. its. polyhedron. and. coordinates. WAT. as. a. ifth. ligand..
This.leads.to.the.formation.of.a.stable.high-energy.intermediate.state.(INT,.12(±2).
kcal.mol
−1
,.Scheme.1.2)..Thus,.the.presence.of.only.one.Zn.equivalent.in.BcII.spe-
cies. leads. to. a. two-step. mechanism. so. that. WAT. replaces. the. nucleophile. during.
the.irst.step.upon.entering.the.Zn1.coordination.shell.and.then.is.activated.in.the.
second.step.as.proton.donor.for.β-lactam.N5.(the.latter.being.the.rate.limiting.step.
of. the. reaction).. In. CcrA,. the. presence. of. Zn2. merges. these. two. movements. in. a.
concerted.single.step—Zn2.already.activates.WAT.as.soon.as.the.OH-Zn1.bond.is.
broken.upon.nucleophilic.attack..This.also.explains.the.improved.catalytic.eficiency.
of.di-Zn.versus.mono-Zn.variants,.as.found.in.the.calculation.of.the.free.energy.of.
activation.for.di-Zn.(Δ
F
= 18(±2).kcal.mol
−1
).and.mono-Zn.(Δ
F
= 21(±3).kcal.mol
−1
).
species,.respectively.
Di-Zn.CcrA.showed.a.highly.concerted.single-step.mechanism.in.which.the.role.
of.the.two.metal.ion.is.crucial.(Figure.1.7,.Scheme.1.2)..In.particular,.the.Zn2.site.
promotes.the.protonation.of.the.bridging.nitrogen.that.kinetic.studies.indicate.to.be.
the.rate-determining.step.
165,180,186,196
.A.series.of.mutagenesis.studies.on.di-Zn.BcII.
conirms.this.view,.since.engineering.a.more.buried.position.of.the.Zn2.site.gives.
rise.to.an.inactive.β-lactamase.even.though.it.is.dinuclear.
197
.Instead,.an.optimized.
BcII.obtained.by.in.vitro.evolution.has.been.shown.to.be.more.eficient.through.the.
action.of.a.second-shell.ligand,.which.optimizes.the.position.of.Zn2.for.the.proton-
ation.step.
198
.These.works.provide.an.excellent.example.of.close.cooperation.between.
theoretical. and. experimental. studies. in. a. metalloenzyme,. and. point. to. an. impor-
tant.and.more.lexible.role.of.the.second.metal.site.in.B1.species..Moreover,.recent.
mechanistic. studies. on. BcII. have. shown. the. accumulation. during. turnover. of. an.
active.mono-Zn.variant.with.the.metal.ion.localized.at.the.DCH.site,.in.contrast.to.
what.was.observed.in.the.irst.reported.x-ray.structure.of.BcII.
199,200
.Unfortunately,.
the.lack.of.structural.data.for.such.conformations.hampers.QM/MM.investigations.
of. this. alternative. pathway,. but. points. consistently. to. the. crucial. role. of. the. Zn2.
metal-binding.site.(Zn1).
Because.the.zinc.ligands.and.most.active.site.residues.are.highly.conserved.among.
subclass.B1.MβLs,.the.binding.and.mechanism.found.for.CcrA.may.be.a.template.for.
the.entire.B1.subclass,.where.β-lactams.might.follow.similar.catalytic.pathways.as.
all.the.groups.involved.in.the.catalysis.do.not.depend.on.the.substrate.chemical.diver-
sity..Of.course,.slightly.different.activation.free.energies.are.expected.because.of.the.
substrate.binding.modes.generated.by.different.β-lactam.substituents.and.differing.
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