A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pK_a of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The K_m^l-Cth of E333 substitution variants is increased ~ 17-fold, while K_m^l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k_cat/K_m^l-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (k_cat/K_m^l-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k_cat/K_m^l-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.     

Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger

Prokaryotic and eukaryotic Na⁺/Ca²⁺ exchangers (NCX) control Ca²⁺ homeostasis. NCX orthologs exhibit up to 10⁴-fold differences in their turnover rates (k_cat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K_m values (K_int = K_m^Cyt/K_m^Ext) are highly asymmetric and alike (K_int ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k_cat at comparable K_int remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k_cat and K_int. Among 55 tested residues, 26 mutations affect either k_cat or K_int, where two major groups can be distinguished. The first group of mutations (14 residues) affect k_cat rather than K_int. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K_int rather than k_cat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca²⁺ and Na⁺ signaling.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

Purification,catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg²⁺ or Mn²⁺, with Mn²⁺ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K_m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k_cat was 69 s⁻¹. The half denaturationn temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.     

Molecular characterization of a thermostable L-fucose isomerase from Dictyoglomus turgidum that isomerizes L-fucose and D-arabinose

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn²⁺ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k_cat of 15,500 min⁻¹ and a K_m of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k_cat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K_m, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k_cat, but had little effect on K_m.     

3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k _cat/K _m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD⁺ over NADP⁺ as a cofactor for its catalytic activity. The k _cat/K _m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD⁺ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k _cat/K _m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ and Ag⁺ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.     

Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

Residues Distal from the Active Site that Alter Enzyme Function in M.HhaI DNA Cytosine Methyltransferase

Abstract

Ten M.HhaI residues were replaced with alanine to probe the importance of distal protein elements to substrate/cofactor binding21,methyl transfer, and product release. The substitutions, ranging from 6–20 Å from the active site were evaluated by thermodynamic analysis, pre-steady and steady-state kinetics, to obtain K_d ^AdoMet, K_d ^DNA, k_cat/K_m ^DNA, k_cat, and k_{methyltransfer} values. For the wild-type M.HhaI, product release steps dominate catalytic turnover while the 4-fold faster internal microscopic constant k_{methyltransfer} presents an upper limit. The methyl transfer reaction has δH? and δS? values of 10.3 kcal/mol and—29.4 cal/(mol K), respectively, consistent with a compressed transition state similar to that observed in the gas phase. Although the ten mutants remained largely unperturbed in methyl transfer, long-range effects influencing substrate/cofactor binding and product release were observed. Positive enhancements were seen in Asp⁷³Ala, which showed a 25fold improvement in AdoMet affinity and in Val²⁸²Ala, which showed a 4-fold improvement in catalytic turnover. Based on an analysis of the positional probability within the C⁵- cytosine DNA methyltransferase family we propose that certain conserved distal residues may be important in mediating long-range effects.     

Cloning, Expression and Characterization of a Trehalose Synthase Gene From Rhodococcus opacus

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K _m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k _cat/K _m. Both 1 and 10 mM of Hg²⁺, Cu²⁺ and Al³⁺ could inhibit the TreS activity, while only 1 mM of Ca²⁺ and Mn²⁺ could increase its activity. Five amino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷ and His³⁵³, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.     

Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase

To evaluate the importance of conserved Arg114 and Arg337 residues of Escherichia coli γ-glutamyltranspeptidase (EcGGT), Lys, Leu, or Asp-substituted mutants were constructed by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 and purified by nickel-chelate chromatography to near homogeneity. With the exception of R114K, all the other mutants significantly lost GGT activity, confirming the importance of these two residues in EcGGT. Kinetic analysis of R114L, R114D, R337K, and R337L revealed a significant increase in K_m with a minor change in k_cat, leading to more than an 8-fold decrease in k_cat/K_m values. Mutations of Arg337 impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments revealed that EcGGT precursor mutants, pro-R337K and pro-R337L, could precede a time-dependent autocatalytic process to generate the small and large subunits, while no autocatalytic processing was observed in pro-R337D. Computer modeling showed that the critical bonding distance of Gln390 O-Thr391 HG1 and Gln390 C-Thr391 OG1 are significantly increased in Arg337 replacements, implying that these distance changes might be responsible for the lack of enzyme maturation.     

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

We used quench flow to study how N⁶-methylated adenosines (m⁶A) affect the accuracy ratio between k_cat/K_m (i.e. association rate constant (k_a) times probability (P_p) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated k_cat/K_m for Glu-tRNA^Glu, EF-Tu and GTP forming ternary complex (T₃) reading cognate (GAA and Gm⁶AA) or near-cognate (GAU and Gm⁶AU) codons. k_a decreased 10-fold by m⁶A introduction in cognate and near-cognate cases alike, while P_p for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated k_cat/K_m for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um⁶AA) and near-cognate (UAG and Um⁶AG) stop codons to decrease 6-fold or 3-fold by m⁶A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m⁶A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate k_cat/K_m, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate k_cat/K_m.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

Characterization of a d-psicose-producing enzyme, d-psicose 3-epimerase, from Clostridium sp.

The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co²⁺ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K _m, turnover number (k _cat), and catalytic efficiency (k _cat/K _m) for d-psicose were 227 mM, 32,185 min^?1, and 141 min^?1 mM^?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l^?1 was produced from 500 g d-fructose l^?1 after 5 h.     

Characterization of a novel thermostable l-rhamnose isomerase from Thermobacillus composti KWC4 and its application for production of d-allose

d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn²⁺ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (K_m), turnover number (k_cat) and catalytic efficiency (k_cat/K_m) for l-rhamnose were 33.8 mM, 1189.8 min⁻¹ and 35.2 min⁻¹ mM⁻¹, respectively. At a higher temperature, Mn²⁺ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (T_m) of T. composti L-RI was increased by 3 °C in presence of Mn²⁺. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI.     

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k_cat/K_m) compared to wild-type CA130. Their K_m values were decreased by ca. 50%, and the k_cat values increased to 14.4 and 16.9 s⁻¹, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.     

Mechanistic insights into F420-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate inhibition studies

F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F₄₂₀ cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F₄₂₀H₂. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F₄₂₀ respectively, thus suggesting a mechanism of ordered addition of substrates in which the F₄₂₀ cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on k_cat and k_cat/K_m for both G6P and F₄₂₀. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d₁ yielded KIE of 1.1 for both k_cat and k_cat/K_m, while the pre-steady state KIE on k_obs was 1.4. Because the hydride transferred to C5 of F₄₂₀ was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²⁺ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K_m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k_cat value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.     

A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg²⁺, Mn²⁺, Ca²⁺, ATP^4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP^4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K _m(MgATP)=1.0 mM,K _i(ATP^4–)=0.27 mM,K _m(MnATP)=0.07 mM, andK _i(CaATP)=0.015 mM. MgATP, MnATP, ATP^4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP^4– from MgATP. Ca²⁺ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK _0.5(Ca²⁺)=0.02 mM,K _0.5(Mn²⁺)=3.8 mM,K _0.5(Mg²⁺)=4.7 mM, and an order of activity Mn²⁺>Mg²⁺>Ca²⁺. The data indicate that Mn²⁺ and Mg²⁺ ions may compete for a regulatory site distinct from the active site and increaseV _m without changingK _m(MgATP),K _m(MnATP), orK _i(ATP^4–). The interactions of ATP^4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg²⁺ and Mn²⁺, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg²⁺ nor Mn²⁺, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP^4– and CaATP are involved in dead-end inhibition. For MgCl₂ saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg²⁺ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg²⁺.Abbreviations used: Me, divalent metal; Me_T (Mg_T or Mn_T), total Me (Me²⁺ and its complexes); ATP_T, total ATP (ATP^4– and its complexes).