Allosteric sites: remote control in regulation of protein activity

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Allosteric regulation of ceruloplasmin activity]

Study of the interactions of homogenous human ceruloplasmin preparations with histamine show that the rate of p-phenylene diamine oxidation by ceruloplasmin is increased in the presence of histamine; the increase in the enzyme activity is independent of histamine concentration. The dependence of the reaction rate on substrate concentration is S-shaped, both in the presence and in the absence of histamine. The respective values of the Hill coefficient and Rs for the enzyme in the presence and in the absence of histamine are 2.5 and 2.0 and 8.0 and 10.4. Histamine does not change ceruloplasmin-specific absorption at 610 nm. Evidence from EPR studies show that histamine does not interact with Cu of the enzyme active center. During interaction with histamine the antigenic properties of the enzyme are changed. Histamine increases the oxidase activity of the enzyme in human and rat blood sera and exerts multifold effects on the enzyme activity in patients with hepatolenticular degeneration. After injection of histamine to rats the enzyme activity is increased without a simultaneous increase in Cu concentration in the blood serum, i.e. without de novo synthesis of ceruloplasmin. The data obtained suggest that ceruloplasmin is probably an allosteric enzyme, which histamine is its positive allosteric effector.     

Allosteric regulation of beef liver arginase activity by L-ornithine

Inhibition of beef liver arginase by L-ornithine was investigated with two sets of independent experiments. Progress curves of the production of urea were simulated with two integrated Michaelis-Menten equations for competitive and non-competitive inhibition by ornithine. Both fitted the curves well, but failed to correctly predict the inhibition when the reaction was started with ornithine already present. Measurement of initial rates of reaction enabled an allosteric model to be built in accordance to Monod-Wyman-Changeux: arginine preferentially binds to the active state R and ornithine preferentially binds to the inactive state T. In the absence of both ligands, the R in equilibrium T equilibrium slightly favours the active state and both states bind ornithine more strongly than arginine. No great variation was observed in the 6 parameters of the model by assuming the enzyme to be a trimer or a tetramer. The model was able to predict not only the initial rate curves, from which it was derived, but also the progress curves independently obtained.     

Allosteric regulation of chaperonins

Chaperonins are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space by complex allosteric regulation. Recently, progress has been made in describing the various functional (allosteric) states of these machines, the pathways by which they interconvert, and the coupling between allosteric transitions and protein folding reactions. However, various mechanistic issues remain to be resolved.     

Extracellular calcium- and magnesium-mediated regulation of passive calcium transport across Caco-2 monolayers

The calcium-sensing receptor (CaR) is expressed on intestinal epithelial serosal membrane and in Caco-2 cells. In renal epithelium, CaR expressed on the basolateral membrane acts to limit excess tubular Ca²⁺ reabsorption. Therefore, here we investigated whether extracellular calcium (Ca_o²⁺) can regulate active or passive ⁴⁵Ca²⁺ transport across differentiated Caco-2 monolayers via CaR-dependent or CaR-independent mechanisms. Raising the Ca_o²⁺ concentration from 0.8 to 1.6 mM increased transepithelial electrical resistance (TER) and decreased passive Ca²⁺ permeability but failed to alter active Ca²⁺ transport. The Ca_o²⁺ effect on TER was rapid, sustained and concentration-dependent. Increasing basolateral Mg²⁺ concentration increased TER and inhibited both passive and active Ca²⁺ transport, whereas spermine and the CaR-selective calcimimetic NPS R-467 were without effect. We conclude that small increases in divalent cation concentration elicit CaR-independent increases in TER and inhibit passive Ca²⁺ transport across Caco-2 monolayers, most probably through a direct effect on tight junction permeability. Whilst it is known that the complete removal of Ca_o²⁺ lowers TER, here we show that Ca_o²⁺ addition actually increases TER in a concentration-dependent manner. Therefore, such Ca_o²⁺-sensitivity could modulate intestinal solute transport including the limiting of excess Ca²⁺ absorption.     

Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.     

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.     

Allosteric regulation of phenylalanine hydroxylase

The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.     

Allosteric regulation of platelet actomyosin

The kinetic properties of platelet actomyosin have been examined to understand the mode of hydrolysis of its substrate ATP. In the presence of divalent cations, ATP hydrolysis deviated from Michaelis-Menten kinetics in such a way as to indicate cooperative effects, with a sigmoidal velocity $\text{vs.}$ substrate curve and a Hill slope of 2.4. In the absence of added divalent cations, linear Michaelis-Menten kinetics were obtained and the Hill slope reduced to 1.0. These results indicate an allosteric regulatory site on platelet actomyosin.     

ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages

The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.