ROLE OF ZINC IN PROTEIN FUNCTION 1 Essential properties of zinc for protein function

Many enzymes and proteins utilize first transition and group DB elements to carry out their biological functions. The most widely used of these metals in biology is zinc (Vallee and Falchuk, 1993). Approximately fifty of the over three hundred enzymes in microorgansms, plants and animals, have been reported to require zinc for their function (Vallee and Falchuk, 1993). The other metalloproteins and enzymes utilize metals including copper, iron, molybdenum, selenium, nickel, manganese and cobalt. The International Union o f Biochemistry has established the categorization of six classes o f enzymes: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases; and zinc can be found in enzymes from all six classes.

Two main properties of zinc are considered to be essential for its wide usage in biology. Firstly, compared with other metals in group HB, zinc is virtually nontoxic (Vallee and Falchuk, 1993). As a result, there is no known disorder associated with the excessive accumulation of zinc which has been reported with iron, copper, mercury and other metals. However, zinc deficiency states are known to result in pathologies of the skin, intestine, nervous systems, reproductive organs and blood. These include growth retardation; high turnover rate of cells from the skin, bowel and gonads; behavioral disorders pertaining to integration of learning, memory and emotional stability; decreased spermatogenesis and reduced output o f testosterone by Ley dig cells in the testes and delayed parturition in pregnant females (Vallee and Falchuk, 1993).

Secondly, the physical and chemical properties of zinc such as stability of association with macromolecules and multiplicity o f co-ordination geometries offer adaptability in the proper function of diverse zinc-containing proteins and enzymes. In a cell, very little zinc is free in solution (Vallee and Falchuk, 1993). Instead, zinc divalent cations are bound to enzymes and proteins. Its resistance to oxidation and reduction invariably offers biological stability in fluctuating cellular environments. At neutrality, zinc is amphoteric, existing as both aquo- and hydoxo-metal complexes. The variabihty in the co-ordination sphere of zinc confers stereochemical adaptability. In other words, this

property enables zinc to assume multiple co-ordination geometries in the zinc-protein complex. Consequently, protein conformation may be determined by the co-ordination geometry adopted. Although two to eight co-ordination geometries are possible with zinc, complexes bearing four, five and six co-ordinate geometries are more frequently encountered. Such geometries result in regular/distorted tetrahedral, trigonal bipyrimidal/square pyrimidal and octahedral symmetries, respectively. Therefore, zinc becomes a versatile interactant for different donor groups on the protein.

1.6.2. Techniques employed in the study of zinc

The recognition o f zinc in biology was a relative late event compared to that o f other essential metabolites. This was attributed to the lack o f suitable analytical methods to detect and quantitate zinc. Early techniques were chemical in nature, with a detection limit of 1 pg/g sample (Vallee and Falchuk, 1993). These early methods were based on the formation of coloured zinc-chelate complexes but was inherently unreliable and relatively insensitive. Present ultrasensitive techniques are based on atomic spectroscopy and the interaction of zinc atoms with electromagnetic radiation. Detection limits have been increased dramatically to lO'^'^g which is about 10*-fold more sensitive than the early chemical method (Vallee and Falchuk, 1993). There are three categories of atomic spectroscopy which are based on atomic emission, atomic absorption and atomic fluoresence. Amongst present techniques, flame atomic absorption spectroscopy is the most widely employed method. The study of zinc has also presently been made possible by new methods for preparing zinc-free buffers and reagents, standards and biological samples for zinc analyses (Falchuk et a l , 1988).

1.6.3. Structure of cysteine-rich domains

The structures of cysteine-rich domains (CRDs) have been well characterized from NMR and X-ray crystallography studies of transcription factors. The first discovery o f a zinc metalloprotein that controlled the transcription o f a specific gene was made by Hanas et al. (1983). This study showed that the Xenopus TFULA transcription factor required for the transcription of the 5S RNA gene by RNA polymerase II was a zinc protein. When the nucleotide sequence o f the gene became available in 1985,

M iller et a l noted that it contained 9 repeats, each of which contained approximately 30 amino acid residues with the -Cys-X2.5-Cys-Xi2.i3-His-X3^-His- sequence. Miller

et al. (1985) then proposed that each of these repeats formed a structure known as a

"zinc finger". Extended X-ray absorption fluoresence spectroscopy (EXAFS) subsequently revealed that one zinc was tetrahedrally co-ordinated to each "zinc finger" via two S and two N atoms as ligands. The number of transcription factors reported to contain the "zinc finger" motif has increased to about 45 proteins (Coleman, 1992). Recent X-ray crystallographic or NM R three-dimensional structural analyses o f other DNA-binding zinc proteins including those of the fungal transcription factor GAL4 (Pan and Coleman, 1990) and the rat glucocorticoid receptor (Luisi et a l , 1991), have led to the recognition of two other distinct motifs in DNA- binding zinc proteins. These are "zinc clusters" in GAL4 and "zinc twist" in the glucocorticoid receptor (Coleman, 1992). In GAL4, two zinc atoms are bound by six cysteines with an interatomic zinc-zinc distance o f 3 Â. Glucocorticoid receptors also contain two zinc atoms, but these are separately bound by four cysteines in each of two distinct sites, leading to an interatomic zinc-zinc distance o f about 13 Â (Luisi et a l , 1991). Hence, these three structures: "zinc finger", "zinc cluster" and "zinc twist" represent standards of reference for the functional domains o f other members o f these protein families.