SH Groups Of Enzyme Research Articles

IPM strategy for rodent control in storage facilities

The fundamental idea of IPM is to put nature?s own regulatory mechanisms to maximum use in order to prevent damage, which is in the best interest of manufacturers, society at large and the environment. However, Haines (2000) assessed stored products IPM still insufficiently developed at the end of the 20th century. Support to IPM development in stored agricultural products is the use of natural compounds to control rodent pests. One of such compounds is cellulose, whose mode of action differs from the modes of other rodenticide active ingredients. As resistance to I and II generation anticoagulant rodenticides has been reported worldwide, we have introduced for the first time anywhere in the world a rodenticide based on Se (0.1% sodium selenite) with a mode of action based on the replacement of SH groups of functional enzymes with SS groups. We also tested products based on vitamin D3 with a known mechanism of action. The new Se-based products achieved good efficacy against rat (91.10 % and 87.50 %) and mouse (97.60% and 98.40%), and cellulose-based product against mouse (91.66%), compared to products based on vitamin D3 in rat (90.00% and 90.30%) and mouse control (98.60% and 98.2%), bromadiolone in rat (93.80% and 90.7%) and mouse control (92.86% and 91.9%) and brodifacoum in mouse control (97.06%). The products based on Se, vitamin D3 and cellulose have good toxicological and ecological properties and the fact that there have been no known cases of resistance make them recommendable for use against rodents in storage facilities.

Purification and properties of mercuric reductase from Azotobacter chroococcum

Mercury resistance determinants in bacteria are often plasmid-borne or transposon-mediated. Mercuric reductase, one of the proteins encoded by the mercury resistance operon, catalyses a unique reaction in which mercuric ions, Hg (II), are reduced to mercury metal Hg(O) using NADPH as a source of reducing power. Mercuric reductase was purified from Azotobacter chroococcum SS2 using Red A dye matrix affinity chromatography. Freshly purified preparations of the enzyme showed a single band on polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS-polyacrylamide gel electrophoresis of the freshly prepared enzyme, two protein bands, a major and a minor one, were observed with molecular weight 69 000 and 54 000, respectively. The molecular weight of the native enzyme as determined by gel filtration in Sephacryl S-300 was 142 000. The Km of Hg2+-reductase for HgCl2 was 11·11 μmol l−1. Titration with 5,5′-dithiobis (2-nitrobenzoate) demonstrated that two enzyme–SH groups become kinetically accessible on reduction with NADPH.

DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.

Inhibitory effect of zinc and copper on phosphatase activity in the brain cytosol of rats: involvement of SH groups.

The effect of various metal ions on neutral phosphatase activity in the brain cytosol of rats was investigated. p-Nitrophenylphosphate was used as the substrate for the enzyme assay. Brain cytosolic phosphatase activity was significantly decreased by the presence of zinc or copper in the range of 10(-5) to 10(-3) M in the enzyme reaction mixture, while it was significantly increased by manganese, nickel or cobalt (10(-5) to 10(-3) M). Calcium (10(-5) to 10(-3) M) did not have an appreciable effect on brain cytosolic phosphatase activity. The inhibitory effect of zinc (10(-5) M) or copper (10(-5) M) on phosphatase activity was seen in the presence of manganese, nickel or cobalt with 10(-4) M concentration, indicating a potent inhibitory effect of these metals. Zinc or copper-decreased phosphatase activity was clearly prevented by dithiothreitol (5 mM), although the effect of each metal was further enhanced by N-ethylmaleimide (5 mM), suggesting that the metals act on the SH-groups of enzyme. Moreover, the inhibitory effect of zinc (10(-5) M) was significantly enhanced by S-100A (10(-6) M) or calbindin (10(-7) M), although neither protein had any effect in the presence of calcium (10(-5) M) or copper (10(-5) M). These results suggests that zinc has an unique role in the regulation of neutral phosphatase activity in rat brain cytosol.

Inhibition of [formula omitted]-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation

In the present work, we studied the effects of phenoxyl radicals, generated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na + K + -ATPase by characterizing interactions of VP-16 phenoxyl radicals with the enzyme's SH-groups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na + K + -ATPase which was dependent on the incubation time and concentration of tyrosinase. The inhibition of Na + K + -ATPase was accompanied by a decrease of DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)-titratable SH-groups. In the presence of Na + K + -ATPase, a typical ESR signal of the VP-16 phenoxyl radical could be observed only following a lag period the duration of which was proportional to the concentration of the Na + K + -ATPase added. Our HPLC measurements demonstrated that Na + K + -ATPase protected VP-16 against tyrosinase-catalyzed oxidation. Combined these results suggest that redox-cycling of VP-16/VP-16 phenoxyl radical by SH-groups of Na + K + -ATPase occurred. Ascorbate which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-groups. Reduction of VP-16 phenoxyl radicals by ascorbate was directly observed by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na + K + -ATPase may be responsible for at least some of its clinical side effects (e.g., cardiotoxicity) which can be prevented by ascorbate.

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

1. 1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents. 2. 2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97. 3. 3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate. 4. 4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The Km for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca2+. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups.

Cisplatin-induced changes in cytochrome P-450, lipid peroxidation and drug-metabolizing enzyme activities in rat kidney cortex

This study investigates the effects of 3 successive cisplatin administrations on rat kidney cytochrome P-450 and drug-metabolizing enzyme activities. Furthermore, because glutathione (GSH) and its related enzymatic system are involved in cellular detoxification processes, we examined the effects of cisplatin on lipid peroxidation, GSH levels, and GSH reductase and peroxidase activities. Cisplatin induced a decrease in cytochrome P-450, GSH, GSH S-transferase, GSH reductase and GSH peroxidase activities, and an increase in N-glucuronyl transferase, lipid peroxidation and oxidized glutathione (GSSG) in kidney cortical microsomes and cytosolic fractions. It is suggested that cisplatin nephrotoxicity could be explained by its affinity for SH-groups of several enzymes and SH-containing compounds. Among these, GSH and its related enzymatic system play a primary role. Moreover, cisplatin increases lipid peroxidation, which might participate in cisplatin nephrotoxicity.

Formation, reactions and toxicity of aldehydes produced in the course of lipid peroxidation in cellular membranes

The formation, reactivity and toxicity of aldehydes originating from the peroxidation of lipids of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals, and particularly 4-hydroxynonenal, are formed in substantial amounts under biological conditions, i.e. during the peroxidation of lipids of liver microsomes incubated in the NADPH-Fe system. Many other aldehydes were then identified in peroxidizing liver microsomes or hepatocytes: alkanals (hexanal the most represented), alk-2-enals and 4-hydroxyalkenals (4-hydroxynonenal and 4,5-dihydroxydecenal, the most represented). The chemical reactivity of the various classes of aldehydes is discussed. Alkenals and 4-hydroxyalkenals are the most reactive aldehydes. The Michael addition to -SH groups of low molecular weight thiols, proteins and enzymes (to give an adduct in which the aldehyde is bound through a thioether linkage to the protein) is by far the main reaction. The in vivo formation of aldehydes in the liver of animals intoxicated with agents that promote lipid peroxidation has been shown in two studies. In the first study, evidence was forwarded for aldehydes (very likely alkenals) bound to the liver microsomal proteins of rats intoxicated with CCl 4 or BrCCl 3. In the second study, 4-hydroxynonenal and a number of other aldehydes (alkanals and alkenals) were identified in liver extracts from bromobenzene-intoxicated mice. The biological implications of the finding of 4-hydroxynonenal and other toxic aldehydes in experimental models of cell injury induced by pro-oxidants are discussed. Finally, recent knowledge on the metabolic pathways for aldehydes, and in particular for 4-hydroxynonenal, is reported.

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5′-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5′-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cystein residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.

Freshly isolated hepatocytes as a model for studying the toxicity of paracetamol

The toxicity of paracetamol has been investigated in freshly isolated hamster hepatocytes. Two phases of toxicity have been identified. In phase 1, metabolic activation of paracetamol occurs with depletion of glutathione. In phase 2, there is progressive morphological damage, leading ultimately to cell death. This occurs even in the absence of further exposure to paracetamol. The thiol reductant, dithiothreitol, added at the start of phase 2, prevents and reverses the toxicological damage that would otherwise occur. Thus, it is most likely that paracetamol causes hepatotoxicity through oxidation of SH groups in key enzymes. N-Acetylcysteine, but not methionine, has an effect similar to that of dithiothreitol. This difference is probably due to oxidation of the enzymes involved in the conversion of methionine to cysteine, whereas N-acetylcysteine can still serve as a precursor of glutathione. The glutathione can act both by adduct formation with the metabolite of paracetamol and as a thiol reductant. Species differences in sensitivity to paracetamol toxicity were shown to be due to differences in the rate of oxidation of the drug to its toxic metabolite. Most people are relatively poor activators of paracetamol, but in a few subjects the reaction proceeds quite rapidly, rendering such individuals more sensitive to the hepatoxic effects of the drug.

Reversal of acetaminophen toxicity in isolated hamster hepatocytes by dithiothreitol

The toxicity of acetaminophen in freshly isolated hamster hepatocytes was investigated. Cells exposed to 2.5 m m acetaminophen for 90 min, followed by washing to completely remove unbound acetaminophen, and resuspension in fresh buffer, showed a dramatic decrease in viability over the ensuing 4.5 hr by which time only 4% of the cells could still exclude trypan blue. During the initial 90-min incubation, there was a substantial depletion of glutathione, to 19% of control values, covalent binding of [ 14C]acetaminophen to cellular proteins, and evidence of morphological changes consistent with some disturbance of the plasma membrane. During subsequent incubation of these cells, covalent binding did not change nor did lipid peroxidation, despite the decrease in viability that occurred. Subsequent incubation of cells exposed to acetaminophen for 90 min in buffer containing 1.5 m m dithiothreitol (DTT), a disulfide-reducing agent, largely prevented the decrease in cell viability and reversed the morphological changes that occurred during the first 90-min incubation. However, there was no change in lipid peroxidation, glutathione content, or covalent binding. It is concluded that acetaminophen interacted with some critical target in the cell, and that this left unchecked, led eventually to the death of the cell. DTT prevented and reversed this effect. The toxicity of acetaminophen, and its reversal by DTT, appear independent of either covalent binding of acetaminophen or lipid peroxidation. In addition, the effect of DTT was independent of the concentration of glutathione, most probably acting by directly reducing oxidized SH-groups in critical enzymes, possibly membrane-bound ATP-dependent Ca 2+ translocases.

Protein Phosphorylation and Translational Control in Reticulocytes: Activation of the Heme-Controlled Translational Inhibitor by Calcium Ions and Phospholipid

The synthesis of globin, the major protein synthesized by reticulocytes, requires the presence of heme, the prosthetic group of hemoglobin. The absence of heme leads to the activation of a nucleotide-independent protein kinase that phosphorylates the alpha subunit of the chain initiation factor eIF-2. This modification interferes with the catalytic function of eIF-2 in protein synthesis initiation. Recent progress in our understanding of the molecular mechanism of this inhibition is briefly reviewed. The same phosphorylation is catalyzed by a different enzyme (DAI) which, while constitutive in reticulocytes, is induced by interferon in other cells. This enzyme is activated by low concentrations of double-stranded RNA in conjunction with ATP. The mechanisms of activation of these enzymes are still poorly understood. HCI is believed to form an inactive complex with heme and become active when the heme is removed by hemoglobin formation. The proinhibitor form of HCI (proHCI) is unstable in vitro and, even in the presence of heme, is irreversibly inactivated by SH-binding reagents, alkaline pH, slightly elevated temperatures, or high hydrostatic pressure. In hemin-supplemented reticulocyte lysates proHCI can also be reversibly activated by oxidized glutathione (GSSG) or NADPH depletion as well as by polyunsaturated fatty acids and by Ca2+-phospholipid. The mechanism of activation of HCI by GSSG has not been clarified although it appears to involve oxidation of proHCI SH groups to disulfides. Like activation by GSSG, the activation of HCI by polyunsaturated fatty acids and by Ca2+-phospholipid also appears to be largely due to oxidation of some of the enzyme's SH groups. There thus appear to be two fully independent mechanisms of HCI activation in reticulocyte lysates, one involving heme deficiency, the other involving oxidation of proHCI SH groups. The latter, but not the former, can be prevented or reversed by NADPH generators or dithiols. ProHCI appears to be maintained in the reduced, inactive state by a system involving NADPH, thioredoxin, and thioredoxin reductase.

Interaction with protein SH groups could be involved in adriamycin cardiotoxicity

The use of the antineoplastic agent adriamycin is limited by its eardiotoxicity. The mechanism of cardiotoxicity has been investigated through the study of adriamycin effects on a number of heart enzymes. Adriamycin inhibited the activity of NADP-isocitrate dehydrogenase, malic enzyme, and 6-phosphogluconate dehydrogenase, three enzymes that have in common the presence of reactive SH groups involved in activity. Adriamyein action was prevented by the presence of proteins or dithioerythrol and mimicked by dithiobis dinitrobenzoate. It is suggested that adriamycin effects are due to interaction with enzyme SH groups by a product of adriamycin metabolism.

Differential sensitivity of brain iodothyronine 5'-deiodinases to sulfhydryl-blocking reagents

Propylthiouracil (PTU) and iodoacetate (IAc) partially inhibit the 5'-deiodination of 3,3',5'-triiodothyronine (rT 3) in cerebral cortex, but PTU has no effect on the 5'-deiodination of thyroxine (T 4). We now report that pretreatment of cerebral cortex microsomes with increasing concentrations of IAc or iodoacetamide inhibits the PTU-sensitive 5'-deiodination of rT 3 with half-maximal inhibition at < 1 μM IAc. PTU-insensitive 5'-deiodination of rT 3 and T 4 requires 100–1000-fold higher concentrations of IAc to achieve similar inhibition. Trypsinization decreases both 5'-deiodinating activities by > 90%. These data suggest that enzyme SH groups do not participate in the 5'-deiodination of T 4 or rT 3 by the PTU-insensitive pathway.

Atypical reaction of ‘essential’ sulfhydryl groups of malic enzyme with 2-nitro-5-thiocyanobenzoate and 2,4-dinitrophenylthiocyanate

Under protective conditions N-ethylmaleimide irreversibly blocks most of the nonessential SH groups of pigeon liver malic enzyme (EC 1.1.1.40) leaving the oxidative decarboxylase activity intact. Reaction between the resultant prereacted enzyme and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) leads to the modification of about seven SH residues/tetramer, of which four fast-reacting groups constitute the ‘essential’ groups responsible for the loss of oxidative decarboxylase activity. 2-Nitro-5-thiocyanobenzoate (NTCB) reacts atypically with the prereacted enzyme by substituting the four ‘essential’ SH residues with one cyano residue and three 2-nitro-5-thiobenzoate residues. The resulting enzyme derivative is 90% inactive. The cyanoenzyme derivative produced by cyanolysis of DTNB-modified prereacted enzyme or NTCB-modified prereacted enzyme has all four ‘essential’ SH groups substituted with cyano groups and possesses half of the original activity. Modification of prereacted enzyme by 2,4-dinitrophenylthiocyanate (DNPT), in contrast to NTCB, results in unequal substitution of the ‘essential’ residues with cyano residues and a single 2,4-dinitrophenyl residue. Dithiothreitol reactivates the DNPT-modified prereacted enzyme by regenerating three ‘essential’ SH residues, but failed to release the dinitrophenyl residue. The atypical reactions of prereacted enzyme with NTCB or DNPT require that the native conformation of the enzyme be retained, since these reagents react by substituting the SH groups of urea-denatured enzyme with only cyano groups.

Interaction of human cytoplasmic aldehyde dehydrogenase E 1 with disulfiram

Two equivalents of symmetrically labeled [ 14C]-disulfiram (tetraethylthiuram disulfide, Antabuse) interact with human liver cytoplasmic aldehyde dehydrogenase (ALDH) E 1 (E.C. 1.2.1.3) inhibiting ca. 90% of total catalytic activity. Inhibition occurs without labeling of the enzyme but is associated with disappearance of four SH groups per molecule of enzyme. Inhibition is reversible by treatment with 2-mercaptoethanol suggesting that disulfiram oxidizes vicinal enzyme SH groups to disulfides. The radioactivity from disulfiram is recovered as diethyldithiocarbamate. SDS gel electrophoresis indicates that vicinal SH groups involved in the interaction with disulfiram occur on the same subunits. This is also consistent with the fact that introduction of disulfide bonds by disulfiram is not accompanied by a major conformational change as evidenced by fluorescence polarization or circular dichroism. Experiments with o-iodosobenzoate suggest the presence of a third SH group in the vicinity of the two interacting with disulfiram. Inhibition of aldehyde dehydrogenase by disulfiram is not easily reversible by glutathione which might explain why new protein synthesis is required to regain enzyme activity in vivo.

Modification of the Cl −-activated arginine aminopeptidases from rat liver and human erythrocytes: A comparative study

The amino acid residues important for the catalytic activity of the Cl −-activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. l-Arginyl- l-phenylalanine and N- l-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme ( Mäkinen and Hopsu-Havu, 1967, Enzymologia 32, 333 ). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl −-activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.

In vitro inhibition of alcohol dehydrogenase by acrylamide: interaction with enzyme-sh groups

Acrylamide, a reactive electrophile, caused a concentration-dependent inhibition of alcohol dehydrogenase (ADH) activity (purified) which was reversed by prior addition of glutathione. Reaction of thiol groups of this enzyme with or/Aophthalaldehyde (OPT), a fluorescent reagent, exhibited characteristic fluorescence maxima at 330 nm excitation and 420 nm emission. Addition of acrylamide to the enzyme resulted in a concentration-dependent (acrylamide and protein) quenching of fluorescence of thiol groups when compared with fluorescence quenching caused by N-ethylmaleimide (NEM), a known thiol ligand. The results demonstrate that acrylamide-induced inhibition of purified ADH activity is mediated through its specific interaction with -SH groups in the enzyme molecule.

Studies with tryptophan metabolites in vitro. effect of zinc, manganese, copper and cobalt ions on kynurenine hydrolase and kynurenine aminotransferase in normal mouse liver

The possible mechanisms by which increasing concentrations of Zn 2+, Mn 2+, Cu 2+, or Co 2+ may affect the vitamin B 6-dependent enzymes kynurenine hydrolase and kynurenine aminotransferase. were studied in normal mouse liver homogenates. It was found that Zn 2+ inhibited kynurenine hydrolase. whereas Mn 2+ activated this enzyme, but both Zn 2+ and Mn 2+ activated kynurenine aminotransferase. Co 2+ and Cu 2+ inhibited both enzymes. This inhibition is attributed to the blocking and inactivation of the -SH groups of these enzymes and may be due to the adequate sequence of the -SH groups in both enzymes for Cu 2+ or Co 2+ action. This is in contrast to the inadequate sequence of these groups in kynurenine aminotransferase for Zn 2+ action. The decreasing order by which these metal ions inhibit (a) kynurenine hydrolase is Cu 2+ > Co 2+ > Zn 2+, and (b) kynurenine aminotransferase is Cu 2+ > Co 2+. The decreasing order of the per cent activation of the aminotransferase enzyme is Mn 2+ > Zn 2+. These decreasing orders fall into a more reasonable order approximating the order of complex stability of these metal ions.