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Negative Cooperativity (negative + cooperativity)
Selected AbstractsKinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyaseFEBS JOURNAL, Issue 14 2002Tadayoshi Kanao The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source] Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesisFEBS JOURNAL, Issue 22 2000Nikolas E. Labrou The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source] REGULATION OF PHENYLALANINE AMMONIA-LYASE ENZYME IN ANNONA FRUIT: KINETIC CHARACTERISTICS AND INHIBITORY EFFECT OF AMMONIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2007R. MALDONADO ABSTRACT In this work, we analyzed the kinetic properties of phenylalanine ammonia-lyase (PAL) extracted from "cherimoya" (Annona cherimola Mill.) fruits ripened at ambient temperature (20C) and stored under several environmental conditions, including high CO2 levels (20%) and low temperature (6C). The effect of different ammonia-related compounds on cherimoya PAL activity was also evaluated. PAL exhibited two different Kmvalues for L-phenylalanine (L-Phe ) and negative substrate cooperativity, with Hill coefficient (napp) values reaching 0.64 and 0.71 for low temperature and high CO2 levels, respectively. The kinetic analysis revealed that ammonia produced mixed inhibition of PAL enzyme, with inhibition constants (Ki and Ki,) values of 0.57 ± 0.2 mM and 2.54 ± 0.2 mM. We propose that the regulation of PAL by ammonia inhibition and the negative cooperativity may be essential in adjusting the active phenylpropanoid metabolism in Annonas to the requirement of L-Phe and in consequence, to the carbon skeleton demand for other anabolic pathways. [source] Thermodynamic Analysis of Receptors Based on Guanidinium/Boronic Acid Groups for the Complexation of Carboxylates, ,-Hydroxycarboxylates, and Diols: Driving Force for Binding and CooperativityCHEMISTRY - A EUROPEAN JOURNAL, Issue 15 2004Sheryl L. Wiskur Dr. Abstract The thermodynamics of guanidinium and boronic acid interactions with carboxylates, ,-hydroxycarboxylates, and diols were studied by determination of the binding constants of a variety of different guests to four different hosts (7,10). Each host contains a different combination of guanidinium groups and boronic acids. The guests included molecules with carboxylate and/or diol moieties, such as citrate, tartrate, and fructose, among others. The Gibbs free energies of binding were determined by UV/Vis absorption spectroscopy, by use of indicator displacement assays. The receptor based on three guanidinium groups (7) was selective for the tricarboxylate guest. The receptors that incorporated boronic acids (8,10) had higher affinities for guests that included ,-hydroxycarboxylate and catechol moieties over guests containing only carboxylates or alkanediols. Isothermal titration calorimetry revealed the enthalpic and entropic contributions to the Gibbs free energies of binding. The binding of citrate and tartrate was investigated with hosts 7,10, for which all the binding events were exothermic, with positive entropy. Because of the selectivity of hosts 8,10, a simple boronic acid (14) was also investigated and determined to be selective for ,-hydroxycarboxylates and catechols over amino acids and alkanediols. Further, the cooperativity of 8 and 9 in binding tartrate was also investigated, revealing little or no cooperativity with 8, but negative cooperativity with 9. A linear entropy/enthalpy compensation relationship for all the hosts 7,10, 14, and the carboxylate-/diol-containing guests was also obtained. This relationship indicates that increasing enthalpy of binding is offset by similar losses in entropy for molecular recognition involving guanidinium and boronic acid groups. [source] A High Performance Theory for Thermodynamic Study on the Binding of Human Serum Albumin with Erbium ChlorideCHINESE JOURNAL OF CHEMISTRY, Issue 2 2009G. Rezaei BEHBEHANI Abstract A thermodynamic study of the interaction between erbium(III) chloride (Er3+) and human serum albumin (HSA) was studied at pH=7.0, 27 and 37 °C in phosphate buffer by isothermal titration calorimetry (ITC). The present study reports the thermodynamic parameters that govern HSA-Er3+ interactions. The extended solvation theory was used to reproduce the enthalpies of HSA-Er3+ interactions over the whole range of Er3+ concentrations. The binding parameters recovered from the new model were attributed to the structural change of HSA and its biological activity. The results obtained indicate that there is a set of two identical binding sites for Er3+ ions with negative cooperativity. The enhancement of complex formation by Er3+ and concomitant increase in ?S suggest that the metal ion plays a role in increasing the number of hydrophobic contacts. The binding parameters discovered from the extended solvation model indicate that the stability of HSA molecule is increased as a result of its interaction with Er3+ ions. [source] | ||||||||||