Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phosphoenolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg²⁺ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co²⁺ > Mn²⁺ > Cd^u2+ > Ni²⁺ ⪢ Fe²⁺ > VO²⁺, while Zn²⁺ and Ca²⁺ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO₂ substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn²⁺ activated enzyme the inhibition was purely non-competitive (K_ii = K_is) towards all substrates, while for the Co²⁺-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn²⁺-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO₃⁻ had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co²⁺-activated enzyme than for its Mn²⁺-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn²⁺ and ADP afforded almost complete protection from inactivation; in the presence of Co²⁺ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.     

Effect of ferric and cupric ions on the inactivation rate of dextransucrase

When ferric ion was added to solutions of the enzyme dextransucrase, first-order followed by second-order inactivation behavior was observed. The initial rapid activity loss was attributed to a ferric ion interacting with the thiol group of the native monomer to form a less active enzyme-ion complex; the second inactivation stage involved enzyme-ion complex aggregation and disulfide cross-link formation. In contrast, Cu²⁺ ion inactivation demonstrated simple first-order kinetics. As with Fe³⁺, Cu²⁺ ions can form complexes with enzyme thiol groups. However, unlike ferric ions, cupric ions can also strongly interact with the imidazole ring of histidine. Since the dextransucrase active site contains two key histidines, imidazole-cupric-ion interactions could potentially inhibit enzymatic activity. Thus, it was hypothesized that first-order Cu²⁺ inactivation kinetics involved the adsorption of this ion to the enzyme's activity site. The addition of a reducing agent such as dithiothreitol can inhibit the second enzyme aggregation stage by breaking disulfide cross-links but cannot restrict the initial formation of metal-enzyme complexes.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

NMR and FT-IR conformational studies of 8-substituted guanine nucleosides and nucleotides and their metal adducts and cancer

NMR and FT-IR Studies of the conformational changes of guanosine and guanosine-5′-monophosphate upon substitution of the H8 of guanine by a heavy, large atom, such as bromine, are presented. The conformational forms, syn, anti, C2′-endo and C3′-endo and gg, gt and tg rotamers of the above molecules are compared to those of their metal (Mg²⁺ and P^t2+) adducts, where the metal is fixed to the N7 nitrogen atom of guanine. The antitumor activity of cisplatin is discussed with relation to the conformational form and the effect of cisplatin is compared to the effects of the Mg²⁺ ion and carcinogens.     

Fungicidal mechanism of chlorine dioxide on Saccharomyces cerevisiae

The fungicidal mechanism of chlorine dioxide on Saccharomyces cerevisiae was investigated. During S. cerevisiae inactivation by ClO₂, protein, DNA, and ion leakage, enzyme activity, genomic DNA structure, and cell ultrastructure were examined. Protein and DNA leakages were not observed, while ion leakages of K⁺, Ca²⁺, and Mg²⁺ were detected and were related to the inactivation rate. The glucose-6-phosphate dehydrogenase, citrate synthase, and phosphofructokinase activities were inhibited and were also correlated with the inactivation rate. Genomic DNA structure was not damaged except for an extremely high ClO₂ concentration (100 mg L^-1). Electron micrographs showed that cell surface damage was pronounced and disruption in inner cell components was also apparent. The ion leakage, the inhibition of key enzyme activities of metabolic pathway, and the alteration of cell structure were critical events in S. cerevisiae inactivation by ClO₂.     

Inactivation of TRPM2 Channels by Extracellular Divalent Copper

Cu²⁺ is an essential metal ion that plays a critical role in the regulation of a number of ion channels and receptors in addition to acting as a cofactor in a variety of enzymes. Here, we showed that human melastatin transient receptor potential 2 (hTRPM2) channel is sensitive to inhibition by extracellular Cu²⁺. Cu²⁺ at concentrations as low as 3 µM inhibited the hTRPM2 channel completely and irreversibly upon washing or using Cu²⁺ chelators, suggesting channel inactivation. The Cu²⁺-induced inactivation was similar when the channels conducted inward or outward currents, indicating the permeating ions had little effect on Cu²⁺-induced inactivation. Furthermore, Cu²⁺ had no effect on singe channel conductance. Alanine substitution by site-directed mutagenesis of His995 in the pore-forming region strongly attenuated Cu²⁺-induced channel inactivation, and mutation of several other pore residues to alanine altered the kinetics of channel inactivation by Cu²⁺. In addition, while introduction of the P1018L mutation is known to result in channel inactivation, exposure to Cu²⁺ accelerated the inactivation of this mutant channel. In contrast with the hTRPM2, the mouse TRPM2 (mTRPM2) channel, which contains glutamine at the position equivalent to His995, was insensitive to Cu²⁺. Replacement of His995 with glutamine in the hTRPM2 conferred loss of Cu²⁺-induced channel inactivation. Taken together, these results suggest that Cu²⁺ inactivates the hTRPM2 channel by interacting with the outer pore region. Our results also indicate that the amino acid residue difference in this region gives rise to species-dependent effect by Cu²⁺ on the human and mouse TRPM2 channels.     

重金属Cd2+和Cu2+胁迫下泥蚶消化酶活性的变化

为了探讨重金属Cd²⁺和Cu²⁺胁迫对泥蚶消化酶活性的影响,运用酶学分析的方法,按《渔业水质标准》（GB 11607）规定的Cd²⁺、Cu²⁺最高限量值的1、2、5、10倍设置重金属离子Cd²⁺、Cu²⁺浓度及其组合,研究了在重金属Cd²⁺、Cu²⁺胁迫下,30d内泥蚶3种消化酶活性的变化规律。结果表明：与空白对照组相比,在重金属Cd²⁺、Cu²⁺或其组合的胁迫下,较低浓度组泥蚶的淀粉酶活性实验前期增强（即被诱导）,实验后期减弱（即被抑制）,较高浓度组泥蚶的淀粉酶活性从实验一开始就减弱,并保持在较低水平,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合Cu²⁺ > （Cd²⁺+Cu²⁺）组合 > Cd²⁺;泥蚶脂肪酶的活性实验前期增强,实验后期转为微减弱或减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺;泥蚶胃蛋白酶的活性实验前期增强,且活性呈现升高-降低-再升高-再降低的变化,实验后期分别表现微增强、微减弱和减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺。可见：环境中的Cd²⁺和Cu²⁺对泥蚶的消化酶活性起着明显的影响作用。     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases

The dimeric Cu–Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu₂Co₂SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co²⁺, Ni²⁺ is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu₂Ni₂SOD1 and acting as a very good mimic of the native Cu ion in Ni₂Zn₂SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini’s paramagnetic NMR pursuits of metalloproteins. We report Ni²⁺ binding to apo wild-type SOD1 and a time-dependent Ni²⁺ migration from the Zn site to the Cu site, and the preparation and characterization of Ni₂Ni₂SOD1, which shows coordination properties similar to those of Cu₂Cu₂SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.     

Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Relationship between infrared spectra and isomorphous substitution in smectites: A computer simulation study

Smectites, members¹ of the 2:1 layer silicate family, share the common feature that two tetrahedral sheets sandwich a sheet of octahedrally coordinated metal ion. The diversity of the members of the 2:1 layer silicates occurs because of their capacity for isomorphous substitution of various cations in the octahedral or tetrahedral sheets. Substitution of a divalent metal ion (such as Mg²⁺) for the trivalent Al³⁺ or a trivalent metal ion (such as Al³⁺) for the tetravalent silicon results in a net negative charge, which then undergoes interaction with positive ions (the exchangeable cations) to form an interlayer hydrated phase. Local density functional (LDF) calculations were employed to model isomorphous substitution of Al³⁺ by Na⁺, K⁺, Mg²⁺, Fe²⁺, and Fe³⁺ in the octahedral layer of a dioctahedral smectite clay such as montmorillonite. The energies of the isomorphous substitution were then compared with the experimental observation. The ordering for successful substitution is Al³⁺ > Fe³⁺ > Mg²⁺ > Fe²⁺ > Na⁺ < K⁺. This ordering is consistent with experimental observation. The vibrational frequencies for the isomorphous substituted systems were calculated by LDF calculation and were compared with the experimental IR results. The results match very well with experiment. This understanding will help in successful prediction of the catalytic activity of smectite clays.     

Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in <Emphasis Type="Italic">Bacillus natto</Emphasis>

Objective

To study the effect of Ca²⁺ on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410.

Results

When the concentration of Ca²⁺ varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH₄Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K _m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl₂ (0.05–0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl₂ in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca²⁺ is an intracellular process.

Conclusion

Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

     

Zinc is the metal cofactor of Borrelia burgdorferi peptide deformylase

Peptide deformylase (PDF, E.C. 3.5.1.88) catalyzes the removal of N-terminal formyl groups from nascent ribosome-synthesized polypeptides. PDF contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (His, His, and Cys) and a water molecule. Previous studies revealed that the metal cofactor is a Fe²⁺ ion in Escherichia coli and many other bacterial PDFs. In this work, we found that PDFs from two iron-deficient bacteria, Borrelia burgdorferi and Lactobacillus plantarum, are stable and highly active under aerobic conditions. The native B. burgdorferi PDF (BbPDF) was purified 1200-fold and metal analysis revealed that it contains ∼1.1 Zn²⁺ ion/polypeptide but no iron. Our studies suggest that PDF utilizes different metal ions in different organisms. These data have important implications in designing PDF inhibitors and should help address some of the unresolved issues regarding PDF structure and catalytic function.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

Thermodynamics of Mg and Ca complexation with AMP,ADP, ATP

The thermodynamic parameters (ΔG, ΔH, ΔS) of complexation have been measured by potentiometric and calorimetric titration for formation of ML and MHL (M  Mg²⁺, Ca²⁺; L  AMP²⁻, ADP³⁻, ATP⁴−). The parameters are interpreted to support a model of inner sphere complexation of the metal cations to the phosphate groups with no evidence of metal-ring interaction in the ML complexes. In the MHL complexes, the protonation (of a ring nitrogen) seemingly leads to ‘backfolding’ interaction between the metal and the ring system in addition to the interaction between the metal and the phosphate groups.