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Results for “PRR proteins”

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PubMed2026

The circadian clock proteins PRR modulate root hair development via the RHD6/RSL module in Arabidopsis.

Root hairs, derived from trichoblasts, are critical for plant growth and environmental adaptation. Although environmental cues are known to influence root hair development, how endogenous timing systems such as the circadian clock integrate into the core transcriptional network governing root hair formation remains unclear. Here, we show that the circadian clock-associated protein PSEUDO-RESPONSE REGULATOR5 (PRR5) physically interacts with ROOT HAIR DEFECTIVE6 (RHD6) and RHD6 LIKE1 (RSL1), two basic helix-loop-helix transcription factors essential for root hair initiation. Genetic analyses suggest that PRR proteins contribute to root hair development under long-day conditions in Arabidopsis thaliana. Simultaneous disruption of PRR5, PRR7, and PRR9 results in defective root hairs, whereas PRR5 overexpression markedly increases root hair density and length. Transcriptomic and RT-qPCR analyses reveal that PRRs enhance the expression of RHD6, RSL1, and multiple downstream root hair-responsive genes, while modulating their temporal expression patterns. Furthermore, PRR5-mediated root hair promotion requires RHD6/RSL1, and PRR proteins enhance RHD6-dependent activation of the RSL4 promoter. PRRs also contribute to root hair development under phosphate-deficient and salt-stress conditions. Together, these findings establish a molecular framework in which PRR proteins regulate the RHD6/RSL network to coordinate root hair development and environmental responses.

Arabidopsis↗

PubMed2025

Identification of BoRR gene family in cauliflower: roles in curd development and salt tolerance.

BACKGROUND: Cauliflower, as an important vegetable crop, the research on its curd formation mechanism and stress-responsive gene networks is of great significance for improving its quality, yield and abiotic stress tolerance. The response regulator (RR) gene family plays a crucial role in the regulation of various life processes of many organisms. In this research, a comprehensive analysis of the BoRR gene family in cauliflower was carried out. RESULTS: A Total of 57 BoRR genes were identified in cauliflower and classified into seven subtypes (type A/B-I/B-II/B-IV/C/B-PRR/Clock PRR) based on sequence homology. Chromosomal mapping showed even distribution across genomes, while physicochemical analysis revealed diverse protein properties (134-915 amino acids, pI 4.51-9.19) with predominant nuclear localization. Structural analyses found all BoRR proteins contain REC-type domains, with subtype-specific features: type A has REC_typeA_ARR, type B harbors REC_typeB_ARR domains, and Clock PRR shows circadian-related psREC_RR domains. Exon numbers range from 2 to 10, with type A BoRR genes having shorter CDS lengths. Collinearity analysis identified 28 pairs of gene duplicates (26 inter-chromosomal). Comparative analysis showed 133 collinear pairs with Brassica napus, 96 with Brassica. rapa, and only 1 with monocots specie (rice and maize). Promoter analysis identified hormone-responsive motifs (ABRE, TGACG), development-related elements (ARE), and stress-responsive sequences (e.g., MBS for drought tolerance) in the promoters of BoRR genes. GO enrichment linked BoRR genes to phosphorelay signaling, cytokinin/ethylene response, and developmental processes like meristem maintenance. Expression profiling during curd development showed type A genes (BoRR23/27/34/38/45) up-regulated in vegetative-reproductive transition, BoRR3/6/12/32/54 in curd enlargement, and several genes like BoRR49 in flower bud differentiation. Salt stress (1.5% NaCl) induced transient expression in 8 of 9 selected BoRR genes at day 1 after treatment. qRT-PCR validated their roles in developmental regulation and salt tolerance. CONCLUSION: This study provides valuable insights into the BoRR gene family in cauliflower, laying a foundation for further understanding its genetic mechanisms and potentially guiding efforts to enhance curd quality and salt tolerance in cauliflower.

Salt Tolerance↗

PubMed1975

Magnetic resonance studies of specificity in binding and catalysis of phosphotransferases.

Two common characteristics of the active site structures of intermediate complexes formed in kinase reactions have been observed by magnetic resonance techniques. First, in creatine, arginine, adenylate and pyruvate kinases (EC2.73.2, 2.7.3.3, 2.7.4.3 and 2.7.1.40, respectively) water is progressively excluded and the structure at the active site is progressively immobilized as each reactant is successively added to the enzyme, as monitored by electron spin resonance (e.s.r) and the enhancement of the proton relaxation rate of water (PRR) due to paramagnetic manganese(II) probe. Significant, and often wide-spread, changes in the protein conformation accompanying successive additions of reaction components are shown with 1H n.m.r. studies of pyruvate kinase. The second characteristic is that, for the ternary enzyme-Mn-nucleotide complexes, two parameters, the e.s.r. spectrum and PRR enhancement values, fall within a range of 10% for all enzymes investigated, with the exception of bovine brain creatine kinase. These similarities suggest a homology in teriary structure at the active sites of these enzymes. An unsuspected aspect of substrate and cofactor specificity has been revealed by e.s.r. spectroscopy of the manganese(II) complexes of the transition-state analogue of creatine kinase (E-MnADP-formate-creatine) and of the ternary phosphoenolpyruvate complex. In the former case, replacement of ADP, the normal substrate, by its substrate analogues IDP or 2acute-deoxyadenosine diphosphate produced two interconvertible species of the transition-state analogue complexes, observed in the e.s.r. spectra as an isotropic species and a highly anisotropic species. With the normal substrate, only the anisotropic species is observed. Similarly, in the case of the complex pyruvate kinase-Mn-phosphoenolpyruvate, when the normal monovalent activator K+ is replaced by the inert tetramethylammonium ion, again two interconvertible species rather than the normal one species are observed by e.s.r. spectroscopy. The implications of these phenomena for the relation of specificity to catalytic efficiency are discussed.

Adenosine Diphosphate↗

PubMed1977

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.

Binding Sites↗

PubMed1975

Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site.

Conformational properties of the active site of formyltetrahydrofolate synthetase from Clostridium cylindrosorum have been examined by EPR spectroscopy and by solvent proton relaxation rate (PPR) studies of manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes give EPR spectra which are very similar to those for the binary Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme MnATP-tetrahydrofolate the EPR line shapes are changed substantially. Spectra for the quaternary complexes exhibit narrow line widths, and the splitting patterns are characteristic of a slightly asymmetric electronic environment for the bound Mn(II). Addition of formate to the ADP quatenary complex induces a further significant narrowing of the EPR line widths, although in the absence of tetrahydrofolate, formate does not influence the EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause changes in the EPR patterns for the higher complexes of the enzyme which involve both ADP and tetrahydololate. However, the Pi effect is not influenced by the presence of formate whereas the characteristic effect of nitrate is potentiated only when formate is present. EPR sectra for the thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p differ significantly from spectra for the binary App(CH)p complex is not influenced by further additions of tetrahydrofolate and of tetrahydorfolate and formate. The failure of spectra for the App(CH)p complex to respond to additions of the other substrates for the reaction is in marked contrast to the behavior found for the natural nucleotide substrates and is tentatively attributed to the lack of a protein-mediated interaction between the nucleotide and tetrahydrofolate binding sites in the analog complex. The frequency dependence of solvent PRR in the presence of the various complexes allows an estimate of the correlation times for electron-nuclear dipolar interaction and thereby the extent of hydration of the bound Mn(II) among the various complexes..

Adenosine Diphosphate↗

PubMed2025

Pan-analysis of intra- and inter-species diversity reveals a group of highly variable immune receptor genes in rice.

Plant immune receptors and their natural variations play a central role in combating disease-causing pathogens. These immune receptors include intracellular nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) and cell-surface pattern recognition receptors (PRRs) that can be further classified as receptor-like proteins (RLPs) and receptor-like kinases (RLKs). Although the NLRome has been characterized, the repertoire and extent of diversity of PRRome remain undetermined in rice. In this study, we examined the diversity of immune receptor genes using high-quality genomes of 309 rice accessions from 8 species within the genus Oryza. A total of 376 310 immune receptor genes were identified, including 149 592 NLR-coding genes and 226 718 PRR coding genes. Shannon entropy analysis revealed a set of immune receptors that display significant intra-species and inter-species diversity in rice. In general, RLPs are more variable than RLKs, while NLRs and LRR-RLPs are more variable than LRR-RLKs. Additionally, NLR and PRR genes exhibit contrasting shoot/root expression patterns, with NLRs generally skewed towards root expression. Furthermore, we found that the size of the LRR-RLK gene families correlates with local annual precipitation, suggesting a stronger selection pressure on LRR-RLK genes in rice accessions grown under wet conditions than dry conditions. In sum, this pan-genomic analysis not only reveals the extensive diversity of the immune receptor repertoires in rice but also provides potential target genes for improving disease resistance in rice.

Oryza↗

PubMed1977

Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase.

Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).

Adenosine Diphosphate↗

PubMed1976

Investigations of equilibrium complexes of myoxin subfragment 1 with the manganous ion and adenosine diphosphate using magnetic resonance techniques.

Electron paramagnetic resonance spectroscopy and water proton relaxation rate (PRR) measurements were used to characterize a complex formed at the myosin subfragment 1 (S1) ATPase site with stoichiometric amounts of Mn(II) and ADP. In the absence of nucleotide, Mn(II) binding at the active site is very weak, although two other classes of sites for Mn(II) on subfragment 1 were identified which are not directly involved in the ATPase reaction. A high affinity Mn(II) site (termed L-site with KL = 3 muM) is associated with a region of the molecule which is susceptible to proteolysis (probably the LC2 light chain subunit) since its stoichiometry depends on the conditions employed for the preparation of subfragment 1 during the papain treatment of myosin. In addition there are a number of weak sites for Mn(II) (termed N-sites) probably associated with anionic groups on the surface of the molecule. In order to study the properties of Mn(II) and ADP bound at the active site by magnetic resonance techniques, subfragment 1 preparations virtually free of the L-site were used, since such an ancillary site competes for the available Mn(II). MnADP binds to subfragment 1 with an apparent dissociation constant, KT, of about 4 muM at 25 degrees. The resultant complex, S1-MnADP, has a low PRR enhancement factor (1.7 at 24.3 MHZ), and its frequency (magnetic field) dependence indicates that this is because there are no readily exchangeable water molecules within the first coordination sphere of Mn(II. Relaxation of the bulk solvent is mediated by protons bound transiently within the outer spheres (4 to 7 A) of the Mn(II). A nitroxide spin label attached to the reactive thiol group of subfragment 1 enhances the solvent PRR, and this property is sensitive to the binding of MgADP to the active site. However, no dipolar spin-spin interaction was detected between the nitroxide group and Mn(II) in the S1-MnADP complex, indicating that the metal ion and thiol group are well separated.

Adenosine Diphosphate↗

PubMed1976

Phospholipase A2 complexes with gadolinium (III) and interaction of the enzyme-metal ion complex with monomeric and micellar alkylphosphorylcholines. Water proton nuclear magnetic relaxation studies.

Gadolinium (III) binds competitively with calcium(II) to porcine pancreatic phospholipase A2 (EC 3.1.1.4) and its zymogen. The enzyme-Gd3+ complex exhibits 4% of the hydrolytic activity of the corresponding Ca2+ complex toward a dispersion of dioctanoyllecithin. Dissociation constants for the Gd3+ complex of enzyme and proenzyme were evaluated from water proton relaxation rate (PRR) titrations. At pH 5.8, the dissociation constants for the Gd3+ complexes of enzyme and zymogen are 0.50 and 0.18 mM, respectively. Dissociation constants for the complexes of enzyme with Ca2+, Eu3+, and Tb3+ were evaluated in PRR titrations by competition of these cations with Gd3+ binding. PRR enhancement factors for the Gd3+ complexes of enzyme and proenzyme are 16.4 and 5.8, respectively, at 22 degrees C and 24.3 MHz. Binding of a homologous series of n-alkylphosphorylcholines to the enzyme-Gd3+ complex was investigated through the influence of monomeric and micellar forms of these amphiphiles on the PRR enhancement factor for the enzyme-bound Gd3+. Separate monomer and micelle binding regions were observed in titrations using n-alkylphosphorylcholines with critical micelle concentrations ranging from 15 muM to 13 mM. In every case, the enhancement factors for the enzyme-Gd3+ complexes were significantly greater than that for the tenary complex, enzyme-Gd3+ -monomer. Morever, a synergism was observed in the binding of Gd3+ and micelles to the enzyme. The magnitudes of the PRR enhancement factors for the enzyme-Gd3+ complexes with micelles of n-alkylphosphorylcholines indicate that the bound Gd3+ is freely accessible to the bulk solvent. These results suggest a model for the enzyme-micelle complex in which the active site is spatially removed from the enzyme-micelle interface.

Animals↗

PubMed1977

Magnetic resonance studies of three forms of creatine kinase. Comparison of the properties of native, CH-S-blocked, and H2NCOCH-blocked enzymes.

Magnetic resonance methods are applied in a comparative study of native creatine kinase from rabbit muscle with two sulfhydryl-modified forms of the enzyme--one inactive form obtained by reaction of the enzyme with iodoacetamide and one form with reduced activity obtained by reaction of the iodoacetamide-sensitive sulfhydryl group with methyl methanethiolsulfonate, which blocks the sulfhydryl with a CH3S-group. Water proton relaxation rate (PRR) titrations with the CH3S-blocked enzyme show that the modification does not alter appreciably the affinities of the enzyme for MnADP and for creatine in the presence of MnADP. Similar measurements for the H2NCOCH2-blocked enzyme indicate that this modification weakens the affinity of the enzyme for MnADP. In agreement with previous findings, there is no observable change in the PRR enhancement upon additions of creatine to solutions of the ternary complex, enzyme-MnADP, for the H2NCOCH2-blocked enzyme. PRR titrations enabled the measurement of binding of creatine to the ternary CH3S-enzyme-MnADP complex and show that specific anions such as nitrate, formate, and thiocyanate decrease the apparent dissociation constant for creatine in its complex with the CH3S-blocked enzyme and MnADP, as is observed with native creatine kinase. However, the change in the PRR enhancement for the CH3S-enzyme-MnADP upon binding of creatine in the presence or absence of anions was appreciably smaller than for the native enzyme. For the H2NCOCH2-blocked enzyme, these anions failed to bring about any influence of creatine on the PRR enhancement. Consistent with the diminished influence of these anions on the PRR enhancement of the CH3S-enzyme-MnADP-creatine complex, EPR spectra of bound Mn(II) show that the CH3S-blocking group interferes with the pronounced anion-induced spectral changes which are observed with the native enzyme. EPR spectra for the H2NCOCH2-enzyme-MnADP complex were not influenced upon additions of creatine, even in the presence of anions. These results suggest that the altered catalytic properties of the CH3S-blocked enzyme arise from structural perturbations at the active site which are also reflected in the PRR enhancement factors and EPR spectral features of the Mn(II) complexes. Moreover, the results clearly indicate that the H2NCOCH2-blocking group, which completely inactivates the enzyme, also eliminates the ability of the MnADP site to sense the presence of the second substrate, creatine, alone and in combination with anions which are structural analogs of the migrating phosphoryl group.

Adenosine Diphosphate↗

PubMed1975

Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme.

Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.

Bacillus↗

PubMed2021

Viral infection of cells within the tumor microenvironment mediates antitumor immunotherapy via selective TBK1-IRF3 signaling.

Activating intra-tumor innate immunity might enhance tumor immune surveillance. Virotherapy is proposed to achieve tumor cell killing, while indirectly activating innate immunity. Here, we report that recombinant poliovirus therapy primarily mediates antitumor immunotherapy via direct infection of non-malignant tumor microenvironment (TME) cells, independent of malignant cell lysis. Relative to other innate immune agonists, virotherapy provokes selective, TBK1-IRF3 driven innate inflammation that is associated with sustained type-I/III interferon (IFN) release. Despite priming equivalent antitumor T cell quantities, MDA5-orchestrated TBK1-IRF3 signaling, but not NFκB-polarized TLR activation, culminates in polyfunctional and Th1-differentiated antitumor T cell phenotypes. Recombinant type-I IFN increases tumor-localized T cell function, but does not mediate durable antitumor immunotherapy without concomitant pattern recognition receptor (PRR) signaling. Thus, virus-induced MDA5-TBK1-IRF3 signaling in the TME provides PRR-contextualized IFN responses that elicit functional antitumor T cell immunity. TBK1-IRF3 innate signal transduction stimulates eventual function and differentiation of tumor-infiltrating T cells.

Animals↗

PubMed1977

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn. Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.

Adenosine Diphosphate↗

PubMed1975

Role of metal ions in Escherichia coli alkaline phosphatase. A study of the metal-water interaction by nuclear relaxation rate measurements on water protons.

The binding of metal to alkaline phosphatase from Escherichia coli and the binding of water and orthophosphate to the Me-2+-enzyme binary complex have been examined by water proton relaxation rate (PRR) measurements. Titration of the three paramagnetic metals, Mn2+, Cu2+, and Co2+, into apoalkaline phosphatase and the titrations of apoenzyme into metal have been carried out. Analysis of the spin-lattice relaxation rates for these titrations and of Scatchard binding curves derived from these results, as well as EPR data, show four tight manganese sites, between two and three tight copper sites, or four cobalt sites per enzyme dimer of molecular weight 80,000. The multiple sites for each metal are indistinguishable by these magnetic resonance techniques. Both the spin-lattice- and spin-spin-relaxation rates exhibit a negative temperature coefficient, showing that these processes are not exchange-limited. From a frequency dependence study of T-1 and from the T-1:T-2 ratio measured at 220 MHz, correlation times from the water-enzyme complexes have been estimated. For H20-Mn-2+-alkaline phosphatase, gamma c equals 1.55 times 10-9 s; for H20-Cu-2+ -alkaline phosphatase, gamma c equals 1.82 times 10-s; and for the cobalt complex, gamma c equals 1.0 times 10-12 s at 4 degrees. Assuming 1 water molecule bound per metal site, these correlation times correspond to the following water-metal distances: gamma (A) is 4.0 A for Mn-2+-H20, 3.4 A for Cu-2+-H20, and 2.8 A for Co-2+-H20. Thus, water is shown to bind directly to the metal atoms of alkaline phosphatase. The correlation between the length of the water-metal bond and the relative activity of the various metalloenzymes support the importance of this binding in the monophosphoesterase reaction catalyzed by alkaline phosphatase. Addition of excess orthophosphate to any of the water-metalloenzyme complexes does not displace an exchangeable water molecule from the metal site. The Mn-PO-4 distance which we have reported earlier (Zukin, R.S., Hollis, D.P., and Gray, G.A. (1973) Biochem. Biophys. Res. Commun. 53, 238) to be 7.3 A is consistent with this finding and suggests a model in which Pi binds to Mn-2+-alkaline phosphatase through a water bridge.

Alkaline Phosphatase↗