Elements and their functions in biological systems Classification of minerals found in the human body. Absorption of minerals, transport of trace elements and normal routes of their exrection. Functions of metal ions in biological systems: ternary complexes of metal ion with substrate and enzyme. mechanism of participation of metal ions in enzymatic catalysis. Characteristics of macroelements and essential microelements. Department of General Chemistry Poznań University of Medical Sciences MD Program

Classification of minerals found in the human body: - macroelements (minerals), required in amounts greater than > 100 mg/day C, H, O, N, S, P, Ca, Mg, Na, K, Cl - microelements (trace elements), required in amounts less than < 100 mg/day essential: Co, Cr, Cu, F, Fe, J, Mn, Mo, Se, Zn probably essential: Ni, Si, Sn, V nonessential: As, B, Cd, Hg, Pb, Sb ….

Relationship between the dosage and the biological effect of elements A – nearly abcence , lethal B, C, D – deficiency of varied degree E – physiological dose F – pharmacological effect G – excess, toxic H – extremely toxic, lethal

Absorption and elimination of minerals and essential trace elements

Transport of trace elements in blood Tb – Transcobalamin Cp – ceruloplasmin Tf – Transferrin a2MG – macroglobin Tg – Thyroglobin Element Albumin Globulins Amino acids Co ++ (Tb) Cr + ++ (Tf) Cu ++ (Cp) Fe J ++ (Tg) Mn ++ (Tf, a2MG) Mo Se Zn + (a2MG)

Normal routes of trace elements exrection Bile Urine Pancreatic juice Sweat Mucosal slough cells Co ++ Cr Cu Fe + Mn Mo Se Zn

Functions of metal ions in biological systems: - catalytic (metalloenzymes, metal-activated enzymes) - structural (hydroxyapatite) - in hormone action (calcium, iodine) - in antioxidants (copper, zinc, manganese, selenium) - as drug components (gold, lithium, platinum)

Catalytic functions of metal ions in biological systems Over 25% of all enzymes contain tightly bound metal ions or require them for activity. Metalloenzymes - contain definite quantity of functional metal ion that is retained throughout purification. Metal ions, most commonly transition metals, are Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+. Metal-activated enzymes - loosely bind metal ions from solution, and require them for catalysis. Usually the presence of the alkali and alkaline earth metal ions is required: Na+, K+, Mg2+, or Ca2+. The distinction between metalloenzymes and metal-activated enzymes thus rests on the affinity of a particular enzyme for its metal ion. The mechanism whereby metal ions perform their functions appear to be similar in metalloenzymes and metal-activated enzymes.

Ternary complexes of metal ion with substrate and enzyme function in catalysis For ternary (3-component) complexes of the catalytic site (Enz), a metal ion (M), and substrate (S) that exhibit 1:1:1 stoichiometry, 4 schemes are possible:

All 4 schemes are possible for metal-activated enzymes. Metalloenzymes cannot form the Enz-S-M complex, because they retain the metal ion throughout purification (ie, are already as Enz-M). Most but not all kinases (ATP:phosphotransferases) form substrate-bridge complexes of the type Enz-nucleotide-M. Phosphotransferases using pyruvate or phosphoenolpyruvate as substrate, enzymes catalyzing other reactions of phosphoenolpyruvate, and carboxylases form metal-bridge complexes. A given enzyme may form one type of bridge complex with one substrate and a different type with another.

ATP4- + M(H2O)62+ ↔ ATP-M(H2O)3 2- + 3 H2O Enzyme-bridge complexes (M-Enz-S): metal ion is presumed to perform structural role maintaining an active conformation or to form a metal bridge to a substrate. The metal ion appears to hold one substrate (ATP) in place and to activate it. Formation of substrate-bridge complexes (Enz-S-M) of nucleoside triphosphates with enzyme, metal, and substrate appears to displace of H2O from the coordination sphere of the metal by ATP: ATP4- + M(H2O)62+ ↔ ATP-M(H2O)3 2- + 3 H2O Enzyme then binds, forming the ternary complex: ATP-M(H2O)32- + Enz ↔ Enz-ATP-M(H2O)32-

Metal-bridge complexes (Enz-M-S) or cyclic complexes are formed at the active site of many carboxypeptidases. It was established that His residue is concerned to form Enz-M binary complexes. ATP – Mg2+ complex

Structural role of metal ions Hydroxyapatite (HA, HAp) - Ca2+, PO43-, Ca10(PO4)6 (OH)2 or [Ca3(PO4)2]3 . Ca(OH)2 99% of calcium in the body is located in bone where it forms with phosphate the hydroxyapatite crystals. HA provides the inorganic and structural component of the skeleton, serves as a large reservoir of calcium and other ions. Fluoride ions react with HA to give hydroxyfluorapatite and fluorapatite, in which OH- ions are replaced by F- , increasing both bone strength and density: Ca10(PO4)6 (OH,F) Ca10(PO4)6 F2

Metal ions in the hormone action calcium iodine Calcium ions mediate some hormonal responses, are essential for blood coagulation, muscle contractility and normal neuromuscular irritability. Ca2+ serves as as intracellular messenger of hormon action. Calmodulin, calcium-dependent hormon is involved in regulating various kinases and enzymes of cyclic nucleotide generation and degradation. Enzymes regulated by Ca2+/calmodulin are: adenylate cyclase, guanylate cyclase, Ca2+-dependent protein kinase, glycogen synthase, phospholipase A2, pyruvate kinase, and many other. Dietary iodine is very efficiently absorbed and transported to the thyroid gland, where it is stored and used for the synthesis of the thyroid hormones triiodothyronine and thyroxine. These hormones play a major role in regulating the metabolic rate of the adult and the growth and development of the child.

Metal ions in antioxidants In superoxide dismutase: copper zinc Cu/Zn-SOD manganese Mn-SOD In glutathione peroxidase: selenium GPx

Metal ions as drug components gold, lithium, platinum

Mechanisms of participation of metal ions in enzymatic catalysis: general acid-base catalysis; metal ions act as a Lewis acid, neutralize negative charge, promote catalysis through charge stabilization or shielding negative charges. Metal ions are often much more effective catalysts than protons because can be present in high concentrations at neutral pH, and can have charges > +1. Metal ions are therefore called „superacids”. covalent catalysis, mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state, approximation of reactants; metal ions bind to substrates to orient them properly for reaction. induction of strain changes in the enzyme or substrate.

Model of the role of Mg2+ as a substrate-bridged complex in the active site of the kinases In hexokinase the terminal phosphate of ATP is transferred to glucose, yielding glucose-6-phosphate. Mg2+ coordinates with ATP to form the true substrate and in addition may stabilize the terminal P-O bond of ATP to facilitate transfer of the phosphate to glucose.

the ferroxidase activity of ceruloplasmin lysyl oxidase Copper ion role in: the ferroxidase activity of ceruloplasmin lysyl oxidase Active site of lysyl oxidase, the enzyme catalyzing cross- link formation in collagen

Molecule of the digestive enzyme carboxypeptidase A contains one Zn2+ ion that is essential for its catalytic action. Zn2+ is coordinated to two nitrogens in histidine side chains and one oxygen in a glutamate side chain.

The role of zinc in the mechanism of reaction of carboxypeptidases Zinc ion bound to the enzyme generates a hydroxyl nucleophile from water molecule, which then attacks the polarized carbonyl of the peptide bond. Zn2+ stabilizes the intermediate product. Intermediate collapses to yield products which disassociate from the enzyme.

Characteristics of macroelements and essential microelements

Calcium distribution in serum Total Ca 2+ 100% ↓ Ca 2+ filtrable Ca 2+ unfiltrable 65% 35% ↓ ↓ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ionized bound with bound with bound with small ligands albumin globulins 53% 12% 28% 7%

Element Serum Whole blood Ca Mg Na K Cu Fe Se Zn Total: 2.1 – 2.6 mmol/L Ionized: 1.0 – 1.3mmol/L 0.75 -1.25 mmol/L (20-25% is protein bound) 136 -145 mmol/L 3.5 – 5.0 mmol/L Men: 11.0 – 22.0 mmol/L Women: 13.4 – 24.4 mmol/L Total: 7.9 – 23.6 mmol/L 0.58 – 1.81 mmol/L 7.6 – 22.9 mmol/L Erythrocytes: 0.8 – 3.3 mmol/L 5.5 - 9.3 mmol/L 0.73 – 3.0 mmol/L 67 – 131 mmol/L ↓ erythrocytes: 87% carbonic anhydrase I - 78% carbonic anhydrase II - 9% SOD - 5.4% Others 7.6% plasma: 9.5% leucocytes: 3.5%