Charge is in charge of enzyme function

【作者】 欧光南； Biyan He；

【Author】 Guangnan Ou;Biyan He;School of Bioengineering,Jimei University;

【机构】 School of Bioengineering,Jimei University；

【摘要】 Understanding the nature and the origin of the catalytic power of enzymes has both fundamental and practical importance.The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts.Theoretically,the most efficient enzymatic reactions should have driving forces from both reactant state and transition state features.We speculate that the enzymatic reaction may undergo three steps:reactant state destabilization,transition state stabilization,and product state destabilization.Then,how can an enzyme do so? Electrostatic interactions play a major role in enzyme catalysis^1,2.Russell and Fersht reported that surface charge changes of subtilisin result in change of activity ³.We can reason,from the electrostatic viewpoint,that the negatively charged species seems to be the catalytically active sites for reactants or products,while the positively charged species for transition states.The repulsive interaction between the electrons in the substrates/products orbitals and the negative electrostatic field destabilizes the system.The energy levels will be increased in negative charge field,while decreased in positive charge field.The cooperation of negative charges and positive charges results in a drastic decrease in the activation energy of a reaction ⁴.In this work,we chose heme-containing catalysts as models.Their peroxidase-like activity at selected temperatures（20-40℃） was determined by the reported method with minor modifications ⁵.20μL of a solution containing horseradish peroxidase(HRP,2.6 mg ml^-1) or myoglobin(Mb,2.0 mg mL^-1) or hemin extract from Mb was added to 1 mL of guaiacol solution(20 mmol L^-1 in 0.2 mol L^-1 phosphate buffer with pH of 6,7,or 8).The reaction was initiated by the addition of 20 μL of 1.00 mol L^-1 H₂ O₂.Formation of colored product was monitored at 470 nm as a function of time.Kinetic data were plotted to determine the initial rates（vo）.From ln（vo）-1/T plot,activation energy（Ea） of guaiacol oxidation by HRP and Mb were compared with that by hemin（Fig.1）.The activation energy decreases in the order of Hemin>Mb >HRP.To explain the above catalytic efficiency,let’s examine the structure of heme active site in HRP and Mb from their crystal structures.The heme prosthetic group is ferriprotoporphyrin IX.Two negatively charged propionate side chains are on the same side of porphyrin plane in HRP（PDB 1 atj,middle-top in Fig.2） while on two sides in Mb（PDB 1 myf,right-top in Fig.2）.Electronic properties of the above hemes were then studied by density functional theory（DFT）.Fig.2 showed that the energy gap between LUMO and HOMO for HRP is much less than that for Mb or hemin,indicating that the heme prosthetic group of HRP tends to lose or gain electrons much easier than that of Mb or hemin.On the basis of the above findings,we may conclude that charge of the heme active site is in charge of enzyme function.更多还原

【Abstract】 Understanding the nature and the origin of the catalytic power of enzymes has both fundamental and practical importance.The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts.Theoretically,the most efficient enzymatic reactions should have driving forces from both reactant state and transition state features.We speculate that the enzymatic reaction may undergo three steps:reactant state destabilization,transition state stabilization,and product state destabilization.Then,how can an enzyme do so? Electrostatic interactions play a major role in enzyme catalysis^1,2.Russell and Fersht reported that surface charge changes of subtilisin result in change of activity ³.We can reason,from the electrostatic viewpoint,that the negatively charged species seems to be the catalytically active sites for reactants or products,while the positively charged species for transition states.The repulsive interaction between the electrons in the substrates/products orbitals and the negative electrostatic field destabilizes the system.The energy levels will be increased in negative charge field,while decreased in positive charge field.The cooperation of negative charges and positive charges results in a drastic decrease in the activation energy of a reaction ⁴.In this work,we chose heme-containing catalysts as models.Their peroxidase-like activity at selected temperatures（20-40℃） was determined by the reported method with minor modifications ⁵.20μL of a solution containing horseradish peroxidase(HRP,2.6 mg ml^-1) or myoglobin(Mb,2.0 mg mL^-1) or hemin extract from Mb was added to 1 mL of guaiacol solution(20 mmol L^-1 in 0.2 mol L^-1 phosphate buffer with pH of 6,7,or 8).The reaction was initiated by the addition of 20 μL of 1.00 mol L^-1 H₂ O₂.Formation of colored product was monitored at 470 nm as a function of time.Kinetic data were plotted to determine the initial rates（vo）.From ln（vo）-1/T plot,activation energy（Ea） of guaiacol oxidation by HRP and Mb were compared with that by hemin（Fig.1）.The activation energy decreases in the order of Hemin>Mb >HRP.To explain the above catalytic efficiency,let’s examine the structure of heme active site in HRP and Mb from their crystal structures.The heme prosthetic group is ferriprotoporphyrin IX.Two negatively charged propionate side chains are on the same side of porphyrin plane in HRP（PDB 1 atj,middle-top in Fig.2） while on two sides in Mb（PDB 1 myf,right-top in Fig.2）.Electronic properties of the above hemes were then studied by density functional theory（DFT）.Fig.2 showed that the energy gap between LUMO and HOMO for HRP is much less than that for Mb or hemin,indicating that the heme prosthetic group of HRP tends to lose or gain electrons much easier than that of Mb or hemin.On the basis of the above findings,we may conclude that charge of the heme active site is in charge of enzyme function.更多还原