Co-operativity in seminal ribonuclease function. Kinetic studies.
Ontology highlight
ABSTRACT:
SUBMITTER: Piccoli R
PROVIDER: S-EPMC1149302 | biostudies-other | 1988 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Generalized microscopic reversibility implies that the apparent rate of any catalytic process in a complex mechanism is paralleled by substrate desorption in such a way that this ratio is held constant within the reaction mechanism [Whitehead (1976) Biochem. J. 159, 449--456]. The physical and evolutionary significances of this concept, for both polymeric and monomeric enzymes, are discussed. For polymeric enzymes, generalized microscopic reversibility of necessity occurs if, within the same reaction sequence, the substrate stabilizes one type of conformation of the active site only. Generalized microscopic reversibility suppresses the kinetic co-operativity of the slow transition model [Ainslie, Shill & Neet (1972) J. Biol. Chem. 247, 7088--7096]. This situation is obtained if the free-energy difference between the corresponding transition states of the two enzyme forms is held constant along the reaction co-ordinate. This situation implies that the 'extra costs' of energy (required to pass each energy barrier) that are not covered by the corresponding binding energies of the transition states vary in a similar way along the two reaction co-ordinates. The regulatory behaviour of monomeric enzymes is discussed in the light of the concept of 'catalytic perfection' proposed by Albery & Knowles [(1976) Biochemistry 15, 5631--5640]. These authors claim that an enzyme will be catalytically 'perfect' when its catalytic efficiency is maximum. If this situation occurs for a monomeric enzyme obeying either the slow transition or the mnemonical model, it can be shown that the kinetic co-operativity disappears. In other words, kinetic co-operativity of a monomeric enzyme is 'paid for' at the expense of catalytic efficiency, and the monomeric enzyme cannot be simultaneously co-operative and catalytically very efficient. This is precisely what has been found experimentally in a number of cases.
| S-EPMC1186138 | biostudies-other
Project description:1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis-Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/nu) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis-Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.
| S-EPMC1183727 | biostudies-other
Project description:A study of the catecholase activity of a latent plant polyphenol oxidase, extracted and purified from the chloroplast membranes of grapes (Vitis vinifera cv. Airen), revealed for the first time a lag phase above pH 5.0, whereas a steady-state rate was reached immediately when pH values were lower, thus suggesting the hysteretic nature of the enzyme. During steady state, the enzyme showed negative co-operativity concomitant with the presence of the lag period, and followed classical Michaelis-Menten kinetics under more acid pH conditions. Statistical analysis of these data showed a minimal value for the extreme Hill coefficient of 0.54 at pH 6.0. This kinetic behaviour of polyphenol oxidase has been interpreted in terms of the pH-induced 'slow' transition mechanism reported by Ricard, Noat & Nari [(1984) Eur. J. Biochem. 145, 311-317] in which the conformational change does not affect the active site of the enzyme.
| S-EPMC1132944 | biostudies-other
Project description:The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn-249-->Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 degrees C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h) approximately 1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (h=2.0-2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively co-operative below 20 degrees C (h approximately 3) and negatively co-operative at 40-50 degrees C (h approximately 0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis-Menten kinetics between 35 and 45 degrees C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 degrees C) and above (h=0.7 at 70-80 degrees C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative behaviour in coenzyme binding under the same experimental conditions used for the wild-type enzyme. In loop 270-275 of the coenzyme domain and segments at the interface of dimer A-B, analyses of the wild-type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme.
| S-EPMC1138974 | biostudies-literature
Project description:3D domain swapping (3D-DS) is a complex protein aggregation process for which no unique mechanism exists. We report an analysis of 3D-DS in bovine seminal ribonuclease, a homodimeric protein whose subunits are linked by two disulfide bridges, based on NMR and biochemical studies. The presence of the covalent bonds between the subunits stabilizes the unswapped dimer, and allows distinct evaluation of the structural and dynamic effects of the swapping with respect to the dimerization process. In comparison with the monomeric subunit, which, in solution has a compact structure without any propensity for local unfolding, both swapped and unswapped dimers show increased flexibility. NMR analysis, together with urea denaturation and hydrogen–deuterium exchange data, indicates that the two dimers have increased conformational fluctuations. Furthermore, we found that the rate-limiting step of both the swapping and unswapping pathways is the detachment of the N-terminal helices from the monomers. These results suggest a new general mechanism in which a dimeric intermediate could facilitate 3D-DS in globular proteins.
| S-EPMC7164040 | biostudies-literature
Project description:The features that distinguish positive from negative co-operativity in double-reciprocal Eadie-Hofstee-Scatchard and Hanes plots, often incorrectly stated to be the sign of curvature or second derivatives, are explained. It is shown how to determine the 'Hill exponent' and interaciton free energies from curves in these plots, and in the simple plot of ligand binding or velocity against free ligand or substrate concentration. New types of plots, where the kind of co-operative behaviour is more obvious than in the traditional ones, are proposed.
| S-EPMC1183983 | biostudies-other
Project description:Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR- and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.
| S-EPMC8319347 | biostudies-literature
Project description:The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.
| S-EPMC1146771 | biostudies-other
Project description:Excitatory synaptic connections in the adult neocortex consist of multiple synaptic contacts, almost exclusively formed on dendritic spines. Changes of spine volume, a correlate of synaptic strength, can be tracked in vivo for weeks. Here, we present a combined model of structural and spike-timing-dependent plasticity that explains the multicontact configuration of synapses in adult neocortical networks under steady-state and lesion-induced conditions. Our plasticity rule with Hebbian and anti-Hebbian terms stabilizes both the postsynaptic firing rate and correlations between the pre- and postsynaptic activity at an active synaptic contact. Contacts appear spontaneously at a low rate and disappear if their strength approaches zero. Many presynaptic neurons compete to make strong synaptic connections onto a postsynaptic neuron, whereas the synaptic contacts of a given presynaptic neuron co-operate via postsynaptic firing. We find that co-operation of multiple synaptic contacts is crucial for stable, long-term synaptic memories. In simulations of a simplified network model of barrel cortex, our plasticity rule reproduces whisker-trimming-induced rewiring of thalamocortical and recurrent synaptic connectivity on realistic time scales.
| S-EPMC6041941 | biostudies-literature
Project description:AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.
| S-EPMC3711915 | biostudies-literature