Coordination dynamics of zinc in proteins.

Publisher Summary This chapter reviews that titration curves do not represent just another way of physically characterizing a protein molecule. More than most other physicochemical methods that are in common use, titration studies tend to emphasize individual differences among proteins, and this is reflected in the chapter. The chapter contains experimental procedures of general utility in the determination of titration data. The foundation for any study of hydrogen ion dissociation in proteins is the electrometric titration curve. To obtain such a curve, one begins with a protein solution of known concentration, at an arbitrary reference pH, adds to it varying amounts of a strong acid or a strong base, and then measures the new pH attained. In a separate experiment, or by means of calculations based on similar experiments, one determines how much acid or base is needed to take a solution, which does not contain protein, but otherwise has the same initial pH, ionic strength, and volume. The difference between the two amounts is the amount of acid or base that is bound to the protein in going from the reference pH to the final pH: a plot of this quantity versus the final pH is the desired titration curve. In plotting this curve, OH - ions bound are counted as H + ions dissociated a procedure, which is always permissible in aqueous solutions.

The Interpretation Of Hydrogen Ion Titration Curves Of Proteins

Metallothionein (MT), despite its high metal binding constant (KZn = 3.2 × 1013 M−1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions—i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total number of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compounds are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asymmetrically involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concentration of other biological chelating agents might well determine the direction of zinc transfer and ultimately affect zinc distribution.

https://www.pnas.org/content/pnas/95/7/3489.full.pdf

Control of zinc transfer between thionein, metallothionein, and zinc proteins

In most plants, exposure to elevated concentrations of heavy metals results in growth inhibition (see Chap. 8). After prolonged metal exposure, sensitive plants develop visible symptoms of toxicity such as chlorosis and necrotic lesions. During the past two and half decades, symptoms of metal toxicity and deficiency have been described extensively (Marschner 1995). However, our knowledge of the biochemical basis of metal toxicity symptoms in plants is still sketchy. It is known that heavy metals can bind to functionally important domains of biomolecules and thereby inactivate them. The result is, for instance, the inhibition of an enzymatic reaction and a disturbance of metabolism (Van Assche and Clijsters 1986). Furthermore, heavy metals have been demonstrated to stimulate formation of free radicals (FR) and reactive oxygen species (ROS), either by direct electron transfer involving metal cations, or as a consequence of metal-mediated inhibition of metabolic reactions (Halliwell and Gutteridge 1984; Elstner 1990). If the plant cell cannot match the increased rate of FR and ROS formation with an increased activity of the antioxidative machinery, uncontrolled oxidation and radical chain reactions will result in oxidative stress. Therefore, the degree of cell damage under heavy-metal stress depends on the rate of FR and ROS formation, and on the efficiency and capacity of detoxification and repair mechanisms. A comparison of closely related metal-sensitive and -tolerant species or ecotypes suggested that an enhanced oxidative defence is not a particular trait conferring heavy-metal tolerance to plants (De Vos et al. 1991). Metal-tolerant plants are usually efficient in avoiding the occurrence of toxic concentrations of of free heavy metal cations in plasmatic compartments, and thereby avoid the development of oxidative stress (Woolhouse 1983).

Free Radicals and Reactive Oxygen Species as Mediators of Heavy Metal Toxicity in Plants

Publisher Summary This chapter discusses the relationship of the three-dimensional structures of bovine carboxypeptidase A (CPA), and of its complexes with substrates and inhibitors, to the functional behavior of this enzyme. In particular, it discusses the basis for substrate specificity, modes of binding, and possible mechanisms of hydrolytic cleavage of substrates for this enzyme. CPA is a zinc-containing proteolytic enzyme, which catalyzes the hydrolysis of carboxy-terminal peptide bonds in protein and peptide substrates. Removal of Zn2+, either by lowering the pH below 5.5 or by the use of a variety of chelating agents at neutral pH, yields an inactive enzyme, apocarboxypeptidase A. Peptidase activity is known for Co2+, Ni2+, Mn2+ and Fe2+ in place of Zn2+, but substitution of Cu2+ for Zn2+ yields an enzyme that is inactive toward all substrates. Esters are also cleaved by CPA and the substitution of Hg2+, Cd2+, or Pb2+ retains esterase activity, although these heavy metal derivatives are not peptidases in solution. However, the crystals of the mercury derivative have shown some peptidase activity.

Carboxypeptidase A: a protein and an enzyme.

Metallocarboxypeptidases: stability constants and enzymatic characteristics.

Apocarboxypeptidase-substrate complexes.

A SERIES of metallocarboxypeptidases has been prepared by the substitution of manganese, cobalt, nickel, copper, cadmium, or mercury for zinc, the element present in and essential to the enzymatic activity of native carboxypeptidase A
1. The stability constants, peptidase and esterase activities of these derivatives have been studied and compared1–3. Zinc is bound to the single sulphydryl group of the apoenzyme4–6, as is cobalt as evidenced by its exchange with zinc and by the spectral changes which accompany its binding2,6. It has been suggested that a sulphur and a nitrogen atom of carboxypeptidase, together with a metal atom, constitute the active centre of the enzyme, their interaction being required for activity5.

Joseph E. Coleman

Papers

Metallocarboxypeptidases: stability constants and enzymatic characteristics.

Apocarboxypeptidase-substrate complexes.

Nitrogen and sulphur at the active centre of carboxypeptidase A.