| The flexible dynamic structure of proteins is significant to the function of proteins,and has been an important research area of many researchers.Infrared spectroscopy is an important tool to detect the ultrafast change of substances.It can recognize the ultrafast conformational changes of proteins at the femtosecond and picosecond levels in time scale and at the molecular atomic scale in spatial resolution.Two-dimensional infrared spectroscopy can provide more conformational information.For a long time,in order to understand the kinetic information of proteins,many scientists have simulated and calculated the infrared spectra of biological macromolecules extensively.The method of electrostatic map is one of the most important methods,and good results have been achieved.Through this method the interaction between solute and solvent is described in different solutions,and the folding of peptide chains and three-dimensional conformation changes of proteins are described in protein system.However,it does not calculate the infrared spectra quantitatively.Each spectrum is obtained by data fitting and only for specific solvents.It did not get good results in solvents with high polarity.Quantum vibrational perturbation method that we developed recently can obtain infrared spectra quantitatively,quickly and accurately.Lactate dehydrogenase(LDH)has been studied for decades.It is a tetramer protein that catalyzes the conversion of pyruvate to lactic acid at four active sites with coenzyme NADH.It is an important enzyme involved in the carbon cycle of biological metabolism.Recently,the conformational heterogeneity of the active center of lactate dehydrogenase has been observed by isotope infrared spectroscopy,which corresponds to the different catalytic rates of the enzyme.Molecular dynamics simulation of human heart lactate dehydrogenase(LDH)has been carried out to determine the linear and two-dimensional Fourier transform infrared(2D-FTIR)spectra for the carbonyl stretch vibration of pyruvate in the tetrameric enzyme,using quantum vibrational perturbation theory.The computed line-shapes of individual subunits are inhomogeneously broadened,and span the entire absorption range of the carbonyl vibration of the full enzyme,indicating the same conformation heterogeneity in the four active sites of LDH.However,each subunit line-shape has different width and peak maximum due to variations in conformation equilibrium in different subunits,corresponding to the spectral multiplets observed experimentally.Since there is a finite time interval before a substrate is converted into products in a given active site,the distribution of such a time coarse-grained average of Michaelis complexes is called active-site heterogeneity.Active-site heterogeneity is distinguished from conformation heterogeneity in that the former is governed by the same energy landscape that gives rise to conformation heterogeneity,but a stochastic enzyme-substrate adduct only samples a particular fraction of the conformation space,limited by the enzyme turn-over and shown as a distribution of waiting times,i.e.,reaction rates,in single-enzyme experiments.The present study showed that different absorption peaks in the C=O stretch region of the Michaelis complex,observed experimentally and reproduced computationally,are due to active-site heterogeneity,as a superposition of the spectral line-shapes of different active sites.Consequently,substates corresponding to these spectral peaks of LDH do not interconvert and they have different reaction rates,as found experimentally.The present active-site heterogeneity mechanism is in complete agreement with the kinetic model derived from isotope-edited infrared and temperature-jump relaxation spectroscopy. |
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