Aeromonas proteolytica aminopeptidase: an investigation of the mode of action using a quantum mechanical/molecular mechanical approach

The aminopeptidase of Aeromonas proteolytica (AAP) belongs to the group of metallo-hydrolases that require two divalent cations for full activity. Such binuclear metal centers are found in several aminopeptidases, raising the question whether a common mechanism, at least partly, is likely. We have used a quantum mechanical/molecular mechanical (QM/MM) approach to investigate the reaction mechanism of AAP. Among several possibilities, one reaction path was found to be clearly the most favorable. Beside the chemical transformation steps, effects of the enzyme environment and the influence of the solvent on the catalytic reaction were included in the study. The results are in good agreement with experimental studies and correspond to a high degree to our previous QM/MM calculations on the reaction mechanism of the related binuclear bovine lens leucine aminopeptidase (blLAP), which, although related to the AAP, has different Zn(2+)-coordination spheres and a different catalytic residue. The mechanisms of the two enzymes as suggested in the literature differ on the mode of coordination of the nucleophile and the identity of the general base. However, the results of this and our previous work on blLAP allow us to identify a common mechanism for the two enzymes. This mechanism is probably quite general for binuclear zinc enzymes.     

Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high‐resolution mass spectrometry

Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design owing to their structural robustness and versatility. Moreover, their characterization provides important insights into protein folding in general. ZnF motifs usually possess an exceptional specificity and high affinity towards Zn(II) ion to drive folding. While the Zn(II) ion is canonically coordinated by two cysteine and two histidine residues, many other coordination spheres also exist in small ZnFs, all having four amino acid ligands. Here we used high‐resolution mass spectrometry to study metal ion binding specificity and primary coordination sphere robustness of a designed zinc finger, named MM1. Based on the results, MM1 possesses high specificity for zinc with sub‐micromolar binding affinity. Surprisingly, MM1 retains metal ion binding affinity even in the presence of selective alanine mutations of the primary zinc coordinating amino acid residues.     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

Metals on the move: zinc ions in cellular regulation and in the coordination dynamics of zinc proteins

Homeostatic control maintains essential transition metal ions at characteristic cellular concentrations to support their physiological functions and to avoid adverse effects. Zinc is especially widely used as a catalytic or structural cofactor in about 3000 human zinc proteins. In addition, the homeostatic control of zinc in eukaryotic cells permits functions of zinc(II) ions in regulation and in paracrine and intracrine signaling. Zinc ions are released from proteins through ligand-centered reactions in zinc/thiolate coordination environments, and from stores in cellular organelles, where zinc transporters participate in zinc loading and release. Muffling reactions allow zinc ions to serve as signaling ions (second messengers) in the cytosol that is buffered to picomolar zinc ion concentrations at steady-state. Muffling includes zinc ion binding to metallothioneins, cellular translocations of metallothioneins, delivery of zinc ions to transporter proteins, and zinc ion fluxes through cellular membranes with the result of removing the additional zinc ions from the cytosol and restoring the steady-state. Targets of regulatory zinc ions are proteins with sites for transient zinc binding, such as membrane receptors, enzymes, protein–protein interactions, and sensor proteins that control gene expression. The generation, transmission, targets, and termination of zinc ion signals involve proteins that use coordination dynamics in the inner and outer ligand spheres to control metal ion association and dissociation. These new findings establish critically important functions of zinc ions and zinc metalloproteins in cellular control.     

Structures of TraI in solution

Cobalt and potassium are biologically important metal elements that are present in a large array of proteins. Cobalt is mostly found in vivo associated with a corrin ring, which represents the core of the vitamin B₁₂ molecule. Potassium is the most abundant metal in the cytosol, and it plays a crucial role in maintaining membrane potential as well as correct protein function. Here, we report a thorough analysis of the geometric properties of cobalt and potassium coordination spheres that was performed with high resolution on a representative set of structures from the Protein Data Bank and complemented by quantum mechanical calculations realized at the DFT level of theory (B3LYP/ SDD) on mononuclear model systems. The results allowed us to draw interesting conclusions on the structural characteristics of both Co and K centers, and to evaluate the importance of effects such as their association energies and intrinsic thermodynamic stabilities. Overall, the results obtained provide useful data for enhancing the atomic models normally applied in theoretical and computational studies of Co or K proteins performed at the quantum mechanical level, and for developing molecular mechanical parameters for treating Co or K coordination spheres in molecular mechanics or molecular dynamics studies.

Structural effects of Zn on cell membranes and molecular models

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1 mM Zn and higher concentrations affect cell membrane and molecular models.     

Zinc supplementation ameliorates glycoprotein components and oxidative stress changes in the lung of streptozotocin diabetic rats

Zinc (Zn) is a component of numerous enzymes that function in a wide range of biological process, including growth, development, immunity and intermediary metabolism. Zn may play a role in chronic states such as cardiovascular disease and diabetes mellitus. Zn acts as cofactor and for many enzymes and proteins and has antioxidant, antiinflammatory and antiapoptotic effects. Taking into consideration that lung is a possible target organ for diabetic complications, the aim of this study was to investigate the protective role of zinc on the glycoprotein content and antioxidant enzyme activities of streptozotocin (STZ) induced diabetic rat tissues. Female Swiss albino rats were divided into four groups. Group I, control; Group II, control + zinc sulfate; Group III, STZ-diabetic; Group IV, diabetic + zinc sulfate. Diabetes was induced by intraperitoneal injection of STZ (65 mg/kg body weight). Zinc sulfate was given daily by gavage at a dose of 100 mg/kg body weight every day for 60 days to groups II and IV. At the last day of the experiment, rats were sacrificed, lung tissues were taken. Also, glycoprotein components, tissue factor (TF) activity, protein carbonyl (PC), advanced oxidative protein products (AOPP), hydroxyproline, and enzyme activities in lung tissues were determined. Glycoprotein components, TF activity, lipid peroxidation, non enzymatic glycation, PC, AOPP, hydroxyl proline, lactate dehydrogenase, catalase, superoxide dismutase, myeloperoxidase, xanthine oxidase, adenosine deaminase and prolidase significantly increased in lung tissues of diabetic rats. Also, glutathione levels, paraoxonase, arylesterase, carbonic anhydrase, and Na⁺/K⁺- ATPase activities were decreased. Administration of zinc significantly reversed these effects. Thus, the study indicates that zinc possesses a significantly beneficial effect on the glycoprotein components and oxidant/antioxidant enzyme activities.     

Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy

X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.     

Physiological and proteomic analyses of <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> under zinc stress

In this study, physiological, biochemical, and proteomic changes of Alternanthera philoxeroides leaves under zinc stress were investigated. Zinc is an essential micronutrient for plants, but it can be toxic at higher concentrations. Accumulations of zinc and MDA in leaves increased significantly with the increase of zinc concentrations. Zn considerably changed the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). Zn also altered the antioxidant level, such as reduced glutathione (GSH) and ascorbic acid (AsA). Therefore, it seems that zinc induced oxidative stress in the leaves of A. philoxeroides, in which we found enhancement of antioxidant enzyme activities and antioxidant concentrations. Protein profiles analyzed by two-dimensional electrophoresis revealed that five protein spots were up-regulated in zinc-treated samples. These differentially displayed proteins were identified by mass spectrometry. The up-regulation of some antioxidant enzymes and stress-related proteins clearly indicated that excess zinc generates oxidative stress that might be disruptive to other important metabolic processes. These results indicate a good correlation between the physiological and biochemical changes in A. philoxeroides leaves exposed to excess zinc. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 546–554. This text was submitted by the authors in English.     

Thionein/metallothionein control Zn(II) availability and the activity of enzymes

Fundamental issues in zinc biology are how proteins control the concentrations of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability constants of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was determined. Its log K is 11.2 +/- 0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8 +/- 0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concentrations. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concentrations of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concentrations of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.     

Picomolar concentrations of free zinc(II) ions regulate receptor protein-tyrosine phosphatase β activity

As key enzymes in the regulation of biological phosphorylations, protein-tyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc(II) ions are known to inhibit these enzymes, but the physiological significance of this inhibition has remained elusive. Employing metal buffering for strict metal control and performing a kinetic analysis, we now demonstrate that zinc(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of the receptor protein-tyrosine phosphatase β (also known as vascular endothelial protein-tyrosine phosphatase). The K(i)((Zn)) value is 21 ± 7 pm, 6 orders of magnitude lower than zinc inhibition reported previously for this enzyme. It exceeds the affinity of the most potent synthetic small molecule inhibitors targeting these enzymes. Inhibition is in the range of cellular zinc(II) ion concentrations, suggesting that zinc regulates this enzyme, which is involved in vascular physiology and angiogenesis. Thus, for some enzymes that are not recognized as zinc metalloenzymes, zinc binding inhibits rather than activates as in classical zinc enzymes. Activation then requires removal of the inhibitory zinc.     

Metal stoichiometry, coenzyme binding, and zinc and cobalt enchange in highly purified yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase, purified from baker's yeast under conditions which exclude contamination by extraneous metal ions, is homogeneous by analytical ultracentrifugation and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a molecular weight of 149,000 as determined by ultracentrifugation time-lapse photography and exhibits specific activities of 430 to 480 U/mg. Zinc analysis by three independent, highly sensitive methods, i.e., atomic absorption spectrometry, atomic fluorescence spectrometry, and microwave-induced plasma emission spectrometry, demonstrates 4 g-atom of catalytically essential Zn per mole of enzyme. No other metal atoms are present in stoichiometrically significant quantities as assessed by emission spectrography. The Stoichiometry of coenzyme binding, 4 mol of NADH/mol of enzyme, is identical to that of zinc, consistent with one coenzyme binding site and one zinc atom per enzyme subunit. Conditions for exchange of the four catalytically essential zinc atoms with ⁶⁵Zn have been developed. These atoms exchange identically under all conditions examined. The resultant radiolabeled enzyme, l(YADH)⁶⁵Zn₄], has the same metal content, specific enzymatic activity, and coenzyme binding properties as the native enzyme. The ⁶⁵Zn of this enzyme serves to monitor the extent and site specificity of cobalt replacement. The fully cobalt-substituted enzyme, [(YADH)Co₄], has a specific activity of 80 U/mg, 17% that of the Zn enzyme, and exhibits absorption and circular dichroic spectra which are consistent with coordination by one or more sulfur ligands in a distorted tetrahedral geometry.     

An insight to conserved water molecular dynamics of catalytic and structural Zn(+2) ions in matrix metalloproteinase 13 of human

Matrix Metalloproteinase (MMP)--13 or Collagenase--3 plays a significant role in the formation and remodeling of bone, tumor invasion and causes osteoarthritis. Water molecular dynamic studies of the five (1XUC, 1XUD, 1XUR, 456C, 830C) PDB and solvated structures of MMP-13 in human have been carried out upto 5 ns on assigning the differential charges (+2, +1, +0.5 e) to both the Zinc ions. The MM and MD-studies have revealed the coordination of three water molecules (W(H), W(I) and W(S)) to Zn(c) and one water to Zn(s). The transition of geometry around the Znc from tetrahedral to octahedral via trigonal bipyramidal, and for Zn(s) from tetrahedral to trigonal bipyramidal are seem interesting. Recognition of two zinc ions through water molecular bridging (Zn(c) - W(H) (W(1))...W(2)....W(3)....H(187) Zn(s)) and the stabilization of variable coordination geometries around metal ions may indicate the possible involvement of Zn(c) ...Zn(s) coupled mechanism in the catalytic process. So the hydrophilic topology and stereochemistry of water mediated coupling between Zn-ions may provide some plausible hope towards the design of some bidentate/polydentate bridging ligands or inhibitors for MMP-13.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

Zinc Transporter Proteins

Zinc, which is involved in the structure of all enzyme classes, is a micro nutrient element and necessary for growth and development. The ability of zinc to function without causing toxic effects is depends on the protection of its homeostasis. Zinc transporter proteins are responsible for keeping zinc at certain concentrations. Based on their predicted membrane topology, Zn transporters are divided into two major families, SLC39s/ZIPs and SLC30s/ZnTs, which transport Zn in opposite directions through cellular and intracellular membranes. ZIPs increases the zinc concentration in the cytosol. For this, the ZIPs carries the zinc from extracellular and intracellular compartments to the cytosol. ZnTs, reduces the concentration of zinc in the cytosol. For this, ZnTs carries the zinc from the cytosol to extracellular and intracellular compartments. After being transported to the cell, 50% of the zinc is found in the cytoplasm, 30–40% in the nucleus, and 10% in the plasma and organelle membranes. The expression of many zinc transporter proteins in the cell is depending on the concentration of zinc and the physiological problems. The aim of this study is to give information about association of zinc transporter proteins with physiological events and health problems.     

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc–sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC₅₀ value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.     

Problems in the measurement of zinc using heparin as an anticoagulant

Although several reports imply that anticoagulants and preservatives contain zinc, the quantity of zinc in heparin, if any, has not been documented. Zinc concentration was determined by flame atomic absorption spectroscopy in varying dilutions of multiple commercially obtained samples of purified sodium heparin N = 15 (microgram Zn/1000 Units heparin). Rubber stoppers of sterile heparin vials and of blood evacuation tubes were incubated in pre-analyzed water or saline on a mechanical shaker with fluid aliquots obtained up to 27 hours and analyzed for zinc content (microgram Zn/0.1 ml). Heparin, with contact or without contact with rubber stoppers, recorded identical zinc concentrations. Zinc concentrations varied from 0.222 +/- 0.01 (mean +/- SE) to 3.49 +/- 0.005 microgram Zn/1000 Units heparin. Leaching of zinc from rubber stoppers of vacutainer tubes (N = 9) was noted only with those containing known chelators of zinc. These results indicate that zinc is present in certain lots of sodium heparin and that caution must be exercised when reporting zinc concentrations of blood samples that contain sodium heparin as the anticoagulant.     

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc-sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC(50) value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.     

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass. In addition, we identify a unique, noncatalytic binding mode for the substrate bicarbonate that occurs in both the H. influenzae and E. coli enzymes. The kinetic and structural analysis indicates that binding of bicarbonate in this site of the enzyme may modulate its activity by influencing a pH-dependent, cooperative transition between active and inactive forms. We hypothesize that the two structural subclasses of beta-CAs may provide models for the proposed active and inactive forms of the H. influenzae and E. coli enzymes.