Assorted Metalloenzymes

Assorted Metalloenzymes • Carbonic anhydrase • Hydration-dehydration reaction • Xylose isomerase • Aldo sugar-keto sugar rearrangement • Arginase • Removal of excess nitrogen • Glutamine synthetase • Incorporation of ammonia • DAHP synthase • Precursor for aromatic acid synthesis • Enolase • Energy production from carbohydrates

Zinc metalloenzymes Functions of Zn-coordinated water Catalytic zinc ligands

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

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 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 changes to a Cys do 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 anhydraserole of zinc generate hydroxide nucleophile nucleophilic attack proton transfer coordinate bicarbonate

Xylose Isomerasemetal 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 Isomerasemetal 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 IsomeraseX-ray structure The enzyme has two separate domains The larger domain contains a binuclear metal ion binding site

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

Arginaserole of histidines arginine + H2O ―> ornithine + urea last step in the urea cycle for excretion of excess nitrogen Effect 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 ۰H126N ºWT ▪H141N Effect on catalytic activity Catalytic activity also decreases in these mutants, even in the presence of high Mn(II) Effect on metal ion binding When treated with a metal ion chelator the WT enzyme retains full activity, while two mutants show drastic activity losses

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

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 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 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 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 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

Enolase 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)

Enolase 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? 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 several 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)

Summary • Carbonic anhydrase uses Zn(II) to generate the hydroxide nucleophile • Xylose isomerase 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 • 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

Assorted Metalloenzymes