Principles of Bioinorganic Chemistry - 2003

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Principles of Bioinorganic Chemistry - 2003
You should have your paper topic approved by
Prof. Lippard this week, if you have not done so
already (by 10/12 please). The oral
presentations will be held in research conference
style at MIT's Endicott House estate in Dedham,
MA, on Saturday, October 18. WEB SITE
web.mit.edu/5.062/www/
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Hydrolytic Enzymes, Zinc and other Metal Ions
PRINCIPLES

• M(OH)n centers supply OH- at pH 7 by lowering
  water pKa
• Mn serves as general Lewis acid, activating
  substrates
• Rate acceleration occurs by internal attack
  within coord. sphere
• Protein side chains greatly assist assembly of
  transition state
• Carboxylate shifts can occur, especially at
  dimetallic centers
• Electrostatic interactions predominate
• Non-redox active metal ions often but not
  universally used

Illustrating the Principles

• Carboxypeptidase, carbonic anhydrase - delivering
  hydroxide
• Alcohol dehydrogenase an oxidoreductase
• Dimetallic metallohydrolases are two metals
  better than one?

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Carboxypeptidase A A Hydrolytic Zinc Enzyme
Reaction catalyzed
RCHC(O)NHR
RCHCO2- NH3R
NH2R
NH2R
Cleaves C-terminal peptide bonds prefers
aromatic residues.
Active site contains a single catalytic zinc,
essential for activity. The glutamate can undergo
a carboxylate shift. Thermolysin has a similar
active site it is an endopeptidase.
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Carboxypeptidase A structure with the inhibitor
glycyl-L-tyrosine bound at the active site. Note
hydrogen bonds to key residues in the active site
that position the substrate moiety for bond
scission.
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Catalytic Mechanism for Carboxypeptidase A
Summary of events 1. Substrate binds orients by
the terminal carboxylate. 2. Deprotonate bound
H2O. 3. Polarize scissile bond by Arg127. 4.
Bound OH- attacks peptide C(O). 5. Form
tetrahedral transition state. 6.Lose 2 peptide
fragments and recycle the enzyme. Principles
illustrated 1. Zinc serves as template. 2.Metal
supplies cleaving reagent, OH-, and organizes key
groups. 3. Chemistry achieved at neutral pH! Kcat
100 s-1 .
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Carbonic Anhydrase, the First Known Zn Enzyme
Reaction catalyzed
CO2 H2O
H2CO3 106 s-1
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Carbonic Anhydrase
PZn(OH2)2
PZn(OH) H
Keq 10-7 M kf/kr
Note Rate 10-2 s-1 at pH 7 kf 106 s-1 in
active site. Paradox The reverse reaction is
diffusion controlled, with kr 1011 M-1 s-1
Thus kf 104 s-1. So how can the turnover be 106
s-1 ? Answer Facilitated diffusion of
protons by buffer components bound to the
enzyme.
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Possible Carbonic Anhydrase Mechanism
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Alcohol Dehydrogenase, an Oxidoreductase
Reaction catalyzed
RCH2 OH NAD
RCHO NADH H
Enzyme contains two 40 kDa polypeptides, each
with 2 Zn2centers in separate domains. One zinc
is structural, the other catalytic.
Catalytic zinc is 20 Å from the surface, near
the nicotinamide binding region. This center is
not required for NAD cofactor binding. Alcohol
substate DO require zinc and bind directly to the
metal center, displacing the coordinated water.
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Schematic Diagram NAD binding to the active
site of LADH, with specific, well-positioned
amino acid side chains holding it in place.
Ethanol is shown bound to the zinc, displacing
water. The system is set to undergo catalysis.
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Note hydride transfers from a-C of alcohol to
nicotinamide ring.
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Alkaline Phosphatase a Dizinc(II) Center
Activates the Substrate
1. The substrate binds to the dizinc center a
nearby Arg also helps activate it. 2. A serine
hydroxyl group attack the phosphoryl group,
cleaving the ester. The phosphate is transferred
to the enzyme, forming a phosphoryl-serine
residue. 3. Hydrolysis of this phosphate ester by
a zinc-bound hydroxide com-pletes the catalytic
cycle. This mechanism is supported by studies
with chiral phosphate esters (ROP18O17O16O)2-
there is no net change in chirality at phoshorus.
1.
3.

2.
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Dimetallics can move the value into the
physiological range near pH 7
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Advantages of Carboxylate-Bridged Dimetallic
Centers in Chemistry and Biology
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Principles illustrated the dimetallic affords
hydroxide the substrate is positioned by
residues in the active site the dimetallic
stabilizes the urea leaving group redox inactive
metal electrostatics
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Metallo-b-lactamases, an Emerging Clinical
Problem
PZn(OH2)2
PZn(OH) H
Keq 10-7M kf/kr
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b-Lactamase from Bacteroides fragilis
N.O. Concha, B.A. Rasmussen, K. Bush, O. Herzberg
(1996), Structure 4, 823-836
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Summary - Points to Remember

• Both mono- and dimetallic centers lower the pKa
  value of bound water, allowing hydroxide to be
  delivered at pH 7.
• Coordination of the leaving group portion of the
  substrate to a metal ion activates the substrate
  for nucleophilic attack.
• Residues not coordinated but in the second
  coordination sphere can participate directly
  (serine in phophatases) or indirectly (arginine
  in alcohol dehydrogenase) in substrate attack,
  orientation, and/or activation.
• Carboxylate shifts facilitate substrate binding,
  activation.
• Redox inactive metal ions (Zn2, Ni2, Mn 2,
  Co2) preferred.