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Assorted Metalloenzymes. Carbonic anhydrase Hydration-dehydration reaction Xylose isomerase Aldo sugar-keto sugar rearrangement Arginase Removal of excess nitrogen AHL hydrolase blocking bacterial cell signaling Glutamine synthetase Incorporation of ammonia into amino acids

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assorted metalloenzymes
Assorted Metalloenzymes
  • Carbonic anhydrase
    • Hydration-dehydration reaction
  • Xylose isomerase
    • Aldo sugar-keto sugar rearrangement
  • Arginase
    • Removal of excess nitrogen
  • AHL hydrolase
    • blocking bacterial cell signaling
  • Glutamine synthetase
    • Incorporation of ammonia into amino acids
  • DAHP synthase
    • Precursor for aromatic acid synthesis
  • Enolase
    • Energy production from carbohydrates
zinc metalloenzymes overview
Zinc metalloenzymesoverview

Functions of Zn-coordinated water

Catalytic zinc ligands

carbonic anhydrase
Carbonic anhydrase

CO2 + H2O <―> HCO3- + H+

Reversible hydration of carbon dioxide to produce bicarbonate

Role – buffering in blood

A bound zinc is located in the center of the enzyme

Acetate binds adjacent to the zinc as a mimic of bicarbonate binding

carbonic anhydrase active site structure
Carbonic anhydraseactive site structure

Zn occupies a 4-coordinate tetrahedral site with three His providing donor atoms

The Zn-coordinated hydroxide is the nucleophile that attacks CO2

The water proton is transferred through a series of bound waters to a flexible histidine acceptor

carbonic anhydrase metal ion binding
Carbonic anhydrasemetal ion binding

Each of the direct ligands to the zinc have been extensively mutated, as well as many of the indirect metal ligands

Mutation of any of the histidine ligands has a drastic effect on zinc binding and on catalysis

Even mutations to Cys does not provide a good binding site for the Zn in carbonic anhydrase

Catalysis and Zn binding are less affected by changes in the indirect ligands

Changing E117 to glutamine causes His119 to tautomerize and alter Zn binding

Thr199 is important to stabilize the Zn-coordinated hydroxide nucleophile

carbonic anhydrase role of zinc
Carbonic anhydraserole of zinc

generate hydroxide


nucleophilic attack

proton transfer

coordinate bicarbonate

xylose isomerase metal ion effects
XyloseIsomerasemetal ion effects

The presence of metal ions has a dramatic effect on the stability of Xylose Isomerases isolated from different organisms

The enzyme from a thermophilic organism is more stable in the absence of metal ions, but its stability is also significantly enhanced in their presence

However, a hyperthermophilic enzyme form is completely stable even in the absence of metal ions

The E. coli and Bacillus enzymes are less stable in the absence of bound metal ions

Addition of metal ions significantly enhances thermostability

Mg(II) is less effective in stabilizing the Bacillus enzyme

FEBS Journal272, 1454 (2005)

xylose isomerase metal ion specificity
XyloseIsomerasemetal ion specificity

Xylose isomerase requires a metal ion for catalytic activity

Different metal ions activate the enzyme from different species to different degrees

Mg(II) is a much more effective activator of the Strep. enzymes, while the Co(II) and Mn(II) forms of Bacillus xylose isomerases are more active

However, at low pH Mg(II) is actually a less effective activation of the Strep. enzymes

Only near neutral pH and above does Mg(II) become a highly effective activator

xylose isomerase x ray structure
Xylose IsomeraseX-ray structure

The enzyme has two separate domains

The larger domain contains a binuclear metal ion binding site

xylose isomerase active site structure
Xylose Isomeraseactive site structure

The metal ions bridge between the enzyme and the sugar substrate

The two Mg(II) ions are coordinated to the enzyme primarily by side chain carboxyl groups

Binding of an inhibitor shows that the metal ions can make numerous interactions with the substrate hydroxyl groups

arginase role of histidines
Arginaserole of histidines

arginine + H2O ―> ornithine + urea

last step in the urea cycle for excretion of excess nitrogen

Effect of mutations on thermal stability

Conservative replacement of two essential histidines causes a decrease in the enzyme stability

Replacement of His141 leads to an increase in thermostability

∆ H101N




Effect of mutations on catalytic activity

Catalytic activity also decreases in these mutants, even in the presence of high Mn(II)

The more stable H141N mutant has the lowest activity

Effect of mutations on metal ion binding

When treated with a metal ion chelator the WT enzyme retains full activity, while two mutants show drastic activity losses

arginase x ray structure
ArginaseX-ray structure

arginase is a trimer with each monomer having a binuclear metal ion binding site

Binuclear manganese cluster

MnA – square pyramidal

MnB – octahedral

Asp124, Asp232 and water bridging ligands

These are the two histidines

that were mutated

ahl hydrolase metal ion coordination
AHL Hydrolasemetal ion coordination

This enzyme hydrolyzes quorum sensing molecules that trigger bacterial virulence

The catalytic activity requires the presence of zinc ions

Mutagenesis studies were carried out to identify the possible metal ion ligands

At least 5 histidines, 3 aspartic acids and a tyrosine seem to play a role in metal ion binding

ahl hydrolase structure
AHL Hydrolasestructure

enzyme with bound HSL

metal ion coordination

As expected from the number of metal ions ligands this enzyme contains a binuclear zinc site

Proc. Natl. Acad. Sci. 102, 17606 (2005)

ahl hydrolase role of metal ions
AHL Hydrolaserole of metal ions

proposed catalytic mechanism

The two Zn ions are bound adjacent to the substrate analogue (HSL)

What is the function of these metal ions ?

1. The Zn ions lower the pK of water to promote hydroxide formation

2. Zn1 and Zn2 stabilize the developing negative charges during hydroxide attack

Proc. Natl. Acad. Sci. 102, 17606 (2005)

glutamine synthetase
Glutamine Synthetase

glutamate + MgATP + NH3 ―> glutamine + MgADP

The enzyme is known to require a Mg(II) ion to bind to ATP

The use of a stable Co(III)-ATP complex showed a requirement from two additional metal ions

These metals were proposed, based on NMR structural studies, to interact with the glutamate and ATP substrates

glutamine synthetase x ray structure
Glutamine synthetaseX-ray structure

Glutamine synthetase is a complex dodecamer composed of two rings of six subunits each

Each subunit contains a binuclear metal ion binding site

glutamine synthetase1
Glutamine Synthetase

The active site channel is located between adjacent subunits (shown in light and dark shading)

The metal ions interact with each of the two substrates, ATP and glutamate, to help facilitate phosphoryl transfer

dahp synthase
DAHP Synthase

erythrose-4-P + PEP ―> DAHP

precursor to aromatic amino acid biosynthesis

Bacteria typically produce three different forms of DAHP synthase, each sensitive to inhibition by one of the aromatic amino acids

Metal content of DAHP synthases

The different enzyme forms have a preference for either Fe or Zn

Metal ion reactivation of DAHP synthases

The highest activity is seen with the Mn-bound enzyme, with somewhat lower activities for the Cd(II) and Fe(II) enzyme forms

dahp synthase1
DAHP Synthase

Kinetics of DAHP synthase (Phe)

The purified Mn-bound enzyme has the highest catalytic activity, although both Fe(II) and Co(II) have higher affinity for this site

UV-visible spectrum

The absence of a strong absorbance between 500-700 nm for the Co(II)-enzyme suggests octahedral coordination geometry

The strong peak at 350 nm in the Cu(II)-enzyme is indicative of a metal-ligand charge transfer band which typically arises from a either a thiolate or an imidazole ligand


DAHP Synthase

structure and metal ion binding

functional enzyme dimer

enzyme monomer

PEP (yellow and orange)

sulfate (green) [E4P site]

Mn(II) (purple sphere)

metal ion ligands:

C87, H369, E411, D441

metal ion coordination

J. Molec. Biol.354, 927 (2005)


2-phosphoglycerate <―> phosphoenolpyruvate

The enzyme can utilize several different divalent metal ions

The Mg(II)-enzyme has the highest activity, but the affinity is higher for Mn(II) and Zn(II)


The structure has been determined with a bound transition state analog

There are two metal ion sites, each interacting with the bound inhibitor

Each Mg(II) binding site contains oxygen donor atoms, from the enzyme, the inhibitor, and from waters

what determines metal ion binding specificity in proteins
What determines metal ion binding specificity in proteins ?

Does the geometric arrangement of protein ligands dictate metal ion specificity?

Or, are the geometric requirements of the metal ion accommodated by protein ligand rearrangements?

Zn-(His)3 sites in a number

of different proteins

It appears that the metal ion will select binding sites in the protein that can best accommodate its geometric and donor atom requirements

Protein Sci.7, 1700 (1998)

  • Carbonic anhydrase uses Zn(II) to generate the hydroxide nucleophile
  • Xyloseisomerase uses two Mg(II) ions to stabilize the enzyme and to provide a binding template for the substrate
  • Arginase uses a binuclear Mn(II) cluster to generate hydroxide while AHL hydrolase uses a binuclear Zn(II) cluster
  • Glutamine synthetase also uses two Mn(II) ions, one to bind to ATP and one to bind the amino acid substrate
  • DAHP synthase can be activated by a variety of divalent metal ions with different catalytic efficiencies
  • Enolase uses two Mg(II) ions to bind to the functional groups of the substrate
  • For Zinc-metalloenzymes the geometry and coordination number of the bound metal ion is dictated primarily by the zinc preferences