Load Balancing Cloud Storage Data Distribution Strategy of Internet of Things Terminal Nodes considering Access Cost.

With the rapid development of Internet of Things (IoT) technology, IoT terminal nodes are facing many challenges in data storage, distribution, and data management. In particular, in the IoT terminal nodes considering access cost, the corresponding data distribution and storage are professional, complex, and miscellaneous. Based on the abovementioned current situation, this article innovatively proposes a complex sensor data placement algorithm based on the cloud storage distribution of IoT terminal nodes. Under this algorithm, the accurate division of IoT data I/O methods is realized through reasonable configuration. Through the adaptive sensing algorithm, while fully considering the access cost of the algorithm, the performance of the IoT data storage system is further optimized. In the corresponding terminal node load balancing problem, this article innovatively proposes the terminal node data sorting and distribution algorithm through the node data. The sorting and distribution algorithm realizes the precise segmentation of the IoT data to be processed, thereby realizing the improvement of data reading and processing speed. Based on the proposed algorithm, this article designs a load balancing cloud storage data distribution optimization system of IoT terminal nodes considering access cost and carries out experimental verification in a real environment. The experimental results show that the data pattern division accuracy corresponding to the proposed distribution strategy is improved to 97.13% and the corresponding data access efficiency is improved to 98.3%, compared with the traditional distribution strategy. Therefore, the data distribution strategy proposed in this article has obvious performance advantages and further promotion value.

The first high pH structure of Escherichia coli aspartate transcarbamoylase.

The activity and cooperativity of Escherichia coli aspartate transcarbamoylase (ATCase) vary as a function of pH, with a maximum of both parameters at approximately pH 8.3. Here we report the first X-ray structure of unliganded ATCase at pH 8.5, to establish a structural basis for the observed Bohr effect. The overall conformation of the active site at pH 8.5 more closely resembles the active site of the enzyme in the R-state structure than other T-state structures. In the structure of the enzyme at pH 8.5 the 80's loop is closer to its position in R-state structures. A unique electropositive channel, comprised of residues from the 50's region, is observed in this structure, with Arg54 positioned in the center of the channel. The planar angle between the carbamoyl phosphate and aspartate domains of the catalytic chain is more open at pH 8.5 than in ATCase structures determined at lower pH values. The structure of the enzyme at pH 8.5 also exhibits lengthening of a number of interactions in the interface between the catalytic and regulatory chains, whereas a number of interactions between the two catalytic trimers are shortened. These alterations in the interface between the upper and lower trimers may directly shift the allosteric equilibrium and thus the cooperativity of the enzyme. Alterations in the electropositive environment of the active site and alterations in the position of the catalytic chain domains may be responsible for the enhanced activity of the enzyme at pH 8.5.

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases.

Carbamoyl phosphate (CP) has a half-life for thermal decomposition of <2 s at 100 degrees C, yet this critical metabolic intermediate is found even in organisms that grow at 95-100 degrees C. We show here that the binding of CP to the enzymes aspartate and ornithine transcarbamoylase reduces the rate of thermal decomposition of CP by a factor of >5,000. Both of these transcarbamoylases use an ordered-binding mechanism in which CP binds first, allowing the formation of an enzyme.CP complex. To understand how the enzyme.CP complex is able to stabilize CP we investigated the mechanism of the thermal decomposition of CP in aqueous solution in the absence and presence of enzyme. By quantum mechanics/molecular mechanics calculations we show that the critical step in the thermal decomposition of CP in aqueous solution, in the absence of enzyme, involves the breaking of the C O bond facilitated by intramolecular proton transfer from the amine to the phosphate. Furthermore, we demonstrate that the binding of CP to the active sites of these enzymes significantly inhibits this process by restricting the accessible conformations of the bound ligand to those disfavoring the reactive geometry. These results not only provide insight into the reaction pathways for the thermal decomposition of free CP in an aqueous solution but also show why these reaction pathways are not accessible when the metabolite is bound to the active sites of these transcarbamoylases.

Time evolution of the quaternary structure of Escherichia coli aspartate transcarbamoylase upon reaction with the natural substrates and a slow, tight-binding inhibitor.

Here, we present a study of the conformational changes of the quaternary structure of Escherichia coli aspartate transcarbamoylase, as monitored by time-resolved small-angle X-ray scattering, upon combining with substrates, substrate analogs, and nucleotide effectors at temperatures between 5 and 22 degrees C, obviating the need for ethylene glycol. Time-resolved small-angle X-ray scattering time courses tracking the T-->R structural change after mixing with substrates or substrate analogs appeared to be a single phase under some conditions and biphasic under other conditions, which we ascribe to multiple ligation states producing a time course composed of multiple rates. Increasing the concentration of substrates up to a certain point increased the T-->R transition rate, with no further increase in rate beyond that point. Most strikingly, after addition of N-phosphonacetyl-l-aspartate to the enzyme, the transition rate was more than 1 order of magnitude slower than with the natural substrates. These results on the homotropic mechanism are consistent with a concerted transition between structural and functional states of either low affinity, low activity or high affinity, high activity for aspartate. Addition of ATP along with the substrates increased the rate of the transition from the T to the R state and also decreased the duration of the R-state steady-state phase. Addition of CTP or the combination of CTP/UTP to the substrates significantly decreased the rate of the T-->R transition and caused a shift in the enzyme population towards the T state even at saturating substrate concentrations. These results on the heterotropic mechanism suggest a destabilization of the T state by ATP and a destabilization of the R state by CTP and CTP/UTP, consistent with the T and R state crystallographic structures of aspartate transcarbamoylase in the presence of the heterotropic effectors.

Use of L-asparagine and N-phosphonacetyl-L-asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase.

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.

N-phosphonacetyl-L-isoasparagine a potent and specific inhibitor of Escherichia coli aspartate transcarbamoylase.

The synthesis of a new inhibitor, N-phosphonacetyl-L-isoasparagine (PALI), of Escherichia coli aspartate transcarbamoylase (ATCase) is reported, as well as structural studies of the enzyme.PALI complex. PALI was synthesized in 7 steps from beta-benzyl L-aspartate. The KD of PALI was 2 microM. Kinetics and small-angle X-ray scattering experiments showed that PALI can induce the cooperative transition of ATCase from the T to the R state. The X-ray structure of the enzyme.PALI complex showed 22 hydrogen-bonding interactions between the enzyme and PALI. The kinetic characterization and crystal structure of the ATCase.PALI complex also provides detailed information regarding the importance of the alpha-carboxylate for the binding of the substrate aspartate.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition.

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side-chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved, small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7 angstroms resolution. Time-resolved, small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side-chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side-chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate-binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low-activity low-affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate-binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.