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Allosteric Regulation (allosteric + regulation)
Selected AbstractsEngineering Allosteric Regulation into Biological CatalystsCHEMBIOCHEM, Issue 18 2009Jacques Fastrez Prof. Abstract Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas. [source] Allosteric Regulation of ProteasesCHEMBIOCHEM, Issue 18 2008Patrick Hauske Abstract Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or small molecule at a site distinct from an enzyme's active center. Allosteric modulators represent an alternative approach to the design and synthesis of small-molecule activators or inhibitors of proteases and are therefore of wide interest for medicinal chemistry. The structural bases of some proteinaceous and small-molecule allosteric protease regulators have already been elucidated, indicating a general mechanism that might be exploitable for future rational design of small-molecule effectors. [source] Functional role of fumarate site Glu59 involved in allosteric regulation and subunit,subunit interaction of human mitochondrial NAD(P)+ -dependent malic enzymeFEBS JOURNAL, Issue 4 2009Ju-Yi Hsieh Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+ -dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and ,KA,Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The KA,Fum and ,KA,Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer,tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer,tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP+ -dependent malic enzyme and cytosolic NADP+ -dependent malic enzyme in fumarate activation and malate cooperativity. [source] The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistanceFEMS MICROBIOLOGY REVIEWS, Issue 2-3 2003Laura S. Busenlehner Abstract The SmtB/ArsR family of prokaryotic metalloregulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of di- and multivalent heavy metal ions. Derepression results from direct binding of metal ions by these homodimeric ,metal sensor' proteins. An evolutionary analysis, coupled with comparative structural and spectroscopic studies of six SmtB/ArsR family members, suggests a unifying ,theme and variations' model, in which individual members have evolved distinct metal selectivity profiles by alteration of one or both of two structurally distinct metal coordination sites. These two metal sites are designated ,3N (or ,3) and ,5 (or ,5C), named for the location of the metal binding ligands within the known or predicted secondary structure of individual family members. The ,3N/,3 sensors, represented by Staphylococcus aureus pI258 CadC, Listeria monocytogenes CadC and Escherichia coli ArsR, form cysteine thiolate-rich coordination complexes (S3 or S4) with thiophilic heavy metal pollutants including Cd(II), Pb(II), Bi(III) and As(III) via inter-subunit coordination by ligands derived from the ,3 helix and the N-terminal ,arm' (CadCs) or from the ,3 helix only (ArsRs). The ,5/,5C sensors Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA, and Mycobacterium tuberculosis NmtR form metal complexes with biologically required metal ions Zn(II), Co(II) and Ni(II) characterized by four or more coordination bonds to a mixture of histidine and carboxylate ligands derived from the C-terminal ,5 helices on opposite subunits. Direct binding of metal ions to either the ,3N or ,5 sites leads to strong, negative allosteric regulation of repressor operator/promoter binding affinity, consistent with a simple model for derepression. We hypothesize that distinct allosteric pathways for metal sensing have co-evolved with metal specificities of distinct ,3N and ,5 coordination complexes. [source] Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenasePROTEIN SCIENCE, Issue 1 2000Yu-Chu Huang Abstract A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2,:1 ,:1 ,. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa , subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the , subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the , subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates , as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase. [source] Purification, crystallization and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, a putative archaeal homologue of ,-AMPKACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Inmaculada Gómez García In mammals, AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (,) and two regulatory subunits (, and ,). The , subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. This work describes the purification and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, an archaeal homologue of ,-AMPK. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for MJ1225 were collected to 2.3,Å resolution using synchrotron radiation. The crystals belonged to space group H32, with unit-cell parameters a = b = 108.95, c = 148.08,Å, , = , = 90.00, , = 120.00°. Preliminary analysis of the X-ray data indicated that there was one molecule per asymmetric unit. [source] Engineering Allosteric Regulation into Biological CatalystsCHEMBIOCHEM, Issue 18 2009Jacques Fastrez Prof. Abstract Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas. [source] | ||||||||||