STUDIES ON EVOLUTION OF IRON AND ZINC CONTAINING ENZYMES

Abstract

Enzymes are biological catalysts which are highly specific with respect to substrate, pH and reaction temperature etc. Enzymes known till date are protein molecules. However, recently some nucleotides have also been found to show catalytic activity. It has been established that one third of all enzymes either contain metals or require metal ions for their biological activity. Metals which are commonly found in living organisms are manganese, iron, cobalt, zinc, molybdenum and copper. One of the rarely studied aspects of enzymes is their possible emergence in biochemical systems. Obviously, the evolution of specific and highly reactive enzymes is not a spontaneous process but under the various chemical and bio logical conditions, millions of minute changes in the primitive structure of enzymes could have taken place which ultimately lead to the perfection and high reactivity of enzymes. Various theories and hypotheses have been put forward considering chemical and biological factors regarding evolution of metalloenzymes. According to Egami, the transition elements in the primeval sea, presumably complexed with the compounds readily available to them and further in the course of chemical evolution, the ligands of the complexes were replaced by biologically important molecules forming early enzymes or proenzymes of broad specificity and conse quently of low activity. The early enzymes formed during chemical evolution kept on modifying with respect to replacement of ligands with more and more biologically appropriate macromolecules reaching their present day form, a complex structure and high specificity and reactivity towards substrates. Beck further extended Egami's hypothesis. In the primitive atmosphere, metals were present in the low oxidation state due to reducing nature of the atmosphere and metal formed complexes with primary ligands like CO, NH,, CN~, (CN)_ and H-,0. Amongst these CN has special significance due to its easy formation under prebiotic conditions and high solubility of cyano complexes. During the (ll) process of chemical evolution, compounds such as amino acids, peptides, carboxylic acids and macrocyclic compounds were formed and are termed as secondary ligands which subsequently replaced primary ligands from metal complexes formed earlier. The metalloenzymes containing iron, zinc, and molybdenum are of special interest due to their widespread occurence in Jiving systems. Also these metals were abundant in primeval sea. The present investigations are therefore, under taken to trace the evolutionary steps of iron and zinc metalloenzymes. The main theme of research work is centralized on the important assumption that mixed ligand cyano complexes of amino acid could have acted as precursors of proenzymes. The results of the investigations have been divided into five chapters. Chapter I gives a general introduction of the topic and literature survey related to metalloenzymes. Various evolutionary hypotheses and theories regarding enzymes and different models proposed by the workers and their relevance to related enzymes have been discussed. Chapter II deals with research methodology comprising the procedural details of the synthesis of complexes and kinetic measurements for catalysed decompo sition of hydrogen peroxide, dehydrogenation of ascorbic acid and NADH, hydrolysis of 4-nitrophenyl acetate and 4-nitrophenyl phosphate. The synthesis of complexes of iron(II) and iron(III) of type [Fe(CN),-(L)]j (where n=2,3 or 4 and L=glycine, histidine, imidazole and triglycine) and zinc complexes of type [Zn(L)?] (where L=glycine, histidine or cysteine) have been briefly discussed. The characterisation of all the above complexes of iron and zinc was done on the basis of I.R. electronic spectra and elemental analysis. Chapter III describes the experimental results related to chemical evolution of peroxidases and catalase. The catalytic activity of iron(II) and iron(III) • (iii) complexes of the type [Fe(CN)5(L)]n~ (where n=2,3 or 4and L=glycine, histidine, imidazole or triglycine) have been tested towards decomposition of hydrogen peroxide. Detail kinetic investigations have been carried out. The order with respect to catalyst has been found to be unity whereas order with respect to H202 is one at lower concentration of H_0 and changes to zero at higher H202 concentrations. The catalytic activity of the complexes was found to be maximum at pH 9.18. The activity of the complexes was found to vary as follows: A. For iron(II) complexes : K^[Fe(CN)6)] <K3[Fe(CN)5(his)]< K^[Fe(CN)5(gly)] < K3[Fe(CN)5(im)] <K^[Fe(CN)5(trigly)]. B. For iron(III) complexes : K3[Fe(CN)6] < K3[Fe(CN)5(trigly)] < K2[Fe(CN)5(his)] < K2[Fe(CN)3(im)] < K^FefCN^gly)]. A tentative mechanism and evolutionary importance of mixed ligand cyano complexes is also discussed. The proposed reaction mechanism for decom position of hydrogen peroxide suggests that [Fe(CN).(OH OOH)]n~ could be the active intermediate and breakdown of this active intermediate is a rate determining step. Rate law was found to be consistent with proposed mechanism. In the primitive environment, the earth's atmosphere was free from molecular oxygen and redox reaction could not have involved the molecular oxygen as such. However, redox reactions could have been carried out by simple compounds specially those containing metals of lower oxidation state. Chapter IV therefore concerns with the evaluation of catalytic activity of mixed ligand complexes of iron(II) for dehydrogenation or ascorbic acid and (iv) NADH both coupled with reduction of methylene blue. The order with respect to ascorbic acid and NADH has been found to be one whereas order with respect to methylene blue is one at lower concentration and changes to zero at higher concentration. In case of catalyst, the order ranges to 0.3-0.5. Mechanism, consistent with kinetic data, have been proposed for dehydro genation reactions. Formation of [Fe(CN)5(HA)]n_ type of species governs the rate of dehydrogenation reaction. Rate law was also found to be in good agreement with proposed mechanism. Chapter V incorporates the results of the work related to the evolution of zinc containing enzymes which are mostly hydrolytic enzymes. The most widely studied hydrolytic enzymes are carboxypeptidase-A and alkaline phosphatase. The common and interesting feature of these enzymes is that atleast two histidyl residues are coordinated at the site of zinc atom. Therefore, amino acid complexes of zinc with glycine, histidine, and cysteine were tested for hydrolysis of 4-nitrophenyl acetate and 4-nitrophenyl phosphate spectrophotometrically. Zinc histidinato complex was found to be catalytically more active towards hydrolysis of 4-NPP. Detail kinetic investigations on catalysed hydro lysis of 4-nitrophenyl acetate and 4-nitrophenyl phosphate were carried out. The effect of pH, catalyst and substrate concentrations on hydrolysis reactions were studied. Alkaline medium is suitable for hydrolysis studies. The order with respect to the catalyst is found as one while that with respect to the substrate is one at lower concentration and tends to zero at higher concentra tion. The tentative mechanisms for the above hydrolytic reactions have been proposed. The breakdown catalyst substrate complex is the rate determining step. The whole work is finally concluded in Chapter VI.