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Carol A Fierke - One of the best experts on this subject based on the ideXlab platform.
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conformational change in the bacillus subtilis rnase p holoenzyme pre trna complex enhances substRate affinity and limits cleavage Rate
RNA, 2009Co-Authors: John Hsieh, Carol A FierkeAbstract:Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5′ maturation of precursor tRNAs. To investigate the mechanism of substRate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNAAsp binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular Rate Constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme–substRate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the Reverse Rate Constant for the conformational change that correlates with the formation of an optimal leader–protein interaction in the RNase P holoenzyme–pre-tRNA complex. Furthermore, the forward Rate Constant for the conformational change becomes Rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pKa in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P•pre-tRNA complex is coupled to the interactions between the 5′ leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.
John Hsieh - One of the best experts on this subject based on the ideXlab platform.
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conformational change in the bacillus subtilis rnase p holoenzyme pre trna complex enhances substRate affinity and limits cleavage Rate
RNA, 2009Co-Authors: John Hsieh, Carol A FierkeAbstract:Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5′ maturation of precursor tRNAs. To investigate the mechanism of substRate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNAAsp binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular Rate Constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme–substRate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the Reverse Rate Constant for the conformational change that correlates with the formation of an optimal leader–protein interaction in the RNase P holoenzyme–pre-tRNA complex. Furthermore, the forward Rate Constant for the conformational change becomes Rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pKa in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P•pre-tRNA complex is coupled to the interactions between the 5′ leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.
M.sc Mathew O Odjighere - One of the best experts on this subject based on the ideXlab platform.
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Equilibrium and Kinetic Studies of the Reaction of Aquomet Derivative of Pigeon Haemoglobin with 5,5 1 Dithiobis (2-Nitrobenzoic Acid)
2020Co-Authors: M.sc Vincent O Akpoveta, Ph.d Osaro K Ize-iyamu, M.sc Osazuwa E Jato, M.sc Mathew O OdjighereAbstract:ABSTRACT The sulphydryl groups Cysf9(93)β and Cysβ5(23)β present in the aquomet derivative of pigeon haemoglobin were investigated for their equilibrium and kinetic parameters by monitoring their reaction with 5,5¹-dithiobis (2-nitrobenzoic acid) (DTNB) with the aid of a UV-visible spectrophotometer. At specific pH (5.7-9.0) the absorbances of mixtures of varying DTNB concentrations (0.015mM-0.34mM) and a fixed concentration of aquomet haemoglobin (50mM haem) thermostated at 25ºC was measured at a wavelength of 412nm. Data was analyzed with the aid of a computer program written on Micromaths scientist software. Equilibrium Constant Ke q varied between 17.3 and 0.02 for both sulphydryl groups reflecting a decrease by almost three orders of magnitude between pH 5.7 and 9.0. The apparent second order Reverse Rate Constant (k r ) for the reaction of the sulphydryl groups with DTNB was calculated to be 26.29 to 63,516 dm Elucidation of the apparent second order Reverse Rate Constant (k r ) which has not been feasible from kinetic experiments was made possible from this study. A quantitative assessment of the pHdependence profile shows that k r increases by almost three orders of magnitude for Cysf9(93)β and two orders of magnitude for Cysβ5(23)β between the pH range studied indicating higher reactivity for Cysf9(93)β. This information is vital for a comprehensive understanding of the kinetics and reactivity of the sulphydryl groups in pigeon haemoglobin (aquomethaemoglobin)
Oyebamiji J Babalola - One of the best experts on this subject based on the ideXlab platform.
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Tertiary conformational transition in sheep hemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate
2020Co-Authors: Kehinde Onwochei Okonjo, Idowu A Adeogun, Oyebamiji J BabalolaAbstract:We have determined the second-order Reverse Rate Constant, k R , for the reaction of 5,5´-dithiobis(2-nitrobenzoate) -DTNB -with sheep hemoglobins as a function of pH from values of the second-order forward Rate Constant, k F , and the equilibrium Constant, K equ , at 25°C: k R ¼ kF Kequ . We demonstRate that (i) inositol hexakisphosphate (inositol-P 6 ) decreases k F and k R by increasing K rt , the r ⇌ t tertiary conformation transition Constant; (ii) the conformation favored for both the forward and Reverse reactions is the r conformation. For stripped hemoglobin we obtain from the k F data a t isomer population of 34.6% (±14) prior to reaction with DTNB; from the k R data we calculate a t isomer population of 44.8% (±4) following reaction with DTNB. In the presence of inositol-P 6 the latter value is increased to 79.5% (±2). These results demonstRate that an allosteric transition occurs on reaction with DTNB and on inositol-P 6 binding
Richard E Waugh - One of the best experts on this subject based on the ideXlab platform.
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micromechanical tests of adhesion dynamics between neutrophils and immobilized icam 1
Biophysical Journal, 2004Co-Authors: Elena B Lomakina, Richard E WaughAbstract:Strong, integrin-mediated adhesion of neutrophils to endothelium during inflammation is a dynamic process, requiring a conformational change in the integrin molecule to increase its affinity for its endothelial counterreceptors. To avoid general activation of the cell, Mg2+ was used to induce the high-affinity integrin conformation, and micromechanical methods were used to determine adhesion probability to beads coated with the endothelial ligand ICAM-1. Neutrophils in Mg2+ bind to the beads with much greater frequency and strength than in the presence of Ca2+. An increase in adhesion strength and frequency was observed with both increasing temperature and contact duration (from 2 s to 1 min, 21 or 37°C). The dependence of adhesion probability on contact time or receptor density yielded estimates of the effective Reverse Rate Constant, kr, and the equilibrium association Constant, Ka, for binding of neutrophils to ICAM-1 coated surfaces in Mg2+: kr ≈ 0.7 s−1 and the product Kaρc ≈ 2.4 × 10−4, where ρc is the density of integrin on the cell surface.