Structural Biochemistry/Enzyme/Effects of pH on enzyme activity
Changes in pH have influence on enzymes. The most favorable pH value is known as the optimum pH. This is the point that the enzyme is most active. This is graphically illustrated in figure.
Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. The optimum pH value will vary greatly from one enzyme to another, as Table shows:
Including temperature and pH there are other factors, such as ionic strength, which can influence the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible.
Enzymes typically are most active in a pH range of 5-9. This is due to the fact that proteins function in an environment that reflects this pH. There are a variety of reasons as to why proteins have a narrow pH range. A variety of amino acid residues as well as the carboxyl and amide termini of proteins have a pKa range in the range of intracellular pH. As a result, a change in pH can protonate or deprotonate a side group, thereby changing its chemical features. For example, carboxyl termini, under deprotonated, could potentially lose an interaction with a adjacent subunit, changing the enzyme conformation. In conclusion, this conformation could cause a decrease in substrate affinity. A more drastic pH change can change the protein folding, thereby completely deactivating the enzyme or cause irreversible proteolysis.
However, pH change can potentially be utilized by enzymes for regulation or protein function. For example, hemoglobin will create a salt bridge when blood plasma is acidic. Therefore, the T-state of hemoglobin is stabilized and have a lower binding affinity to oxygen. This facilitates increased oxygen transport to oxygen-deficient muscles.
1. The binding of the substrate to enzyme.
2. The ionization states that the amino acid residues of the catalytic site of the enzyme have.
3. The ionization state of the substrate.
4. The variation in protein structure (More significant at extreme pH values).
The rates of many enzymatic reactions adhere to a bell shaped curve when they are a function of pH:
These curves reflect the ionization state of the amino acid residues that must have a specific ionization state for enzymatic activity to take place. The observed pK's (maxima point) often hints at the identity of the amino acid residues which are essential for enzymatic activity. For instance, an observed pK of ~4 suggests that either an Asp of a Glu is essential to the enzyme. pK of ~6 can hint towards a His residue whereas pK of ~10 hints toward a Lys residue.
However, it is crucial to remember that the micro-environment in which the enzyme is in also affects its activity. For example, an Asp residue in a non-polar environment or in close proximity to another Asp residue would attract protons more strongly than in any other environment and this have a higher pK value.
Moreover, pH effects on an enzyme could cause denaturation of the enzyme rather than protonation or deprotonation of specific catalytic residues.
A particular residue may be replaced by doing site directed mutagenesis. Doing so provides researchers with a reliable approach to identifying residues that are required for substrate binding or catalysis.
Specific Case: The Bohr Effect
The Bohr Effect was named after Christian Bohr, who studied and discovered the effects of Hydrogen ion and Carbon dioxide. The discovery of the cooperativity of Hemoglobin has helped Bohr in studying the effects of pH in enzymes. In this specific case, this is focused on the effect of Hydrogen ions on the hemoglobin protein and enzyme. Before understanding the Bohr effect, the cooperativity of hemoglobin has to be explained. In hemoglobin's cooperativity, the release of oxygen is favored when they are at a high concentration of oxygen. This occurs because the special hemoglobin character facilitate oxygen binding when one active site binds to a oxygen first. This ability of hemoglobin will allow them to respond to other physiological signals at where more oxygen is needed.
In this case, high rates of metabolizing tissue at contracting muscles usually generate high rates of hydrogen ions and carbon dioxide, which allosteric effectors at that bind to hemoglobin on the areas that are not oxygen-binding sites. The Bohr effect is the when the hydrogen and carbon dioxide regulate the oxygen-bindings site on hemoglobin.
As we know that hydrogen ion decreases the pH values in a solution, and this phenomenon usually decreases the hemoglobin's affinity for oxygen, in other word, it increases the release of oxygen. Thus, at high pH, the side chains of the histidine (Beta-146) is not protonated and the salt bridge is not formed, while at low pH the side chains of histidine does form salt bridges when they are protonated. This will result in stabilization of the T state in hemoglobin which also increase the release of oxygen.
When Carbon Dioxide is passed through the human body, the first mechanism that happens is when they react with water to become carbonic acid (H2CO3) accelerated by carbonic anhydrase. Carbonic acid is readily dissociated into HCO3- and H+ and decrease the value of pH as it is described in the previous mechanism.
Another way that Carbon dioxide can affect the affinity of oxygen in hemoglobin is a direct mechanism of Carbon dioxide and hemoglobin. Carbon dioxide stabilizes the deoxyhemoglobin (T state) by reacting with the terminal amino groups to form carbamate groups (negative charged). These carbamate groups then are free to form salt bridges that stabilizes the T state and release oxygen. The formation of the carbamate groups is catalyzed by carbonic anhydrase. After the formation of the carbamate groups, the carbamate dissociates into bicarbonate ions and protons. The salt bridges are formed by protonating histidine which then bridges with asparagine.
Effect of Temperature on Enzymatic Activity
As all enzymes have an optimal pH in which their catalytic activity is at its peak, enzymes also have an optimal temperature. There are two established thermal properties of enzymes that effect the catalytic rate. Those two are activation energy and their thermal stability. However experimental data of temperature against enzymatic activity does not clearly match the so sought out for indignation that activity simply increases with temperature. A new model called the Equilibrium model helps provide the quantitative explanation of enzyme thermal behavior under reaction conditions by introducing the enzymes inactive form, forming an equilibrium system that would follow similar rules to Le Chatlier's principle in basic chemistry. The equilibrium model gives rise to a number of insights in the sense that it eliminates time dependency that was thought to be important in the classical view of enzymatic activity against temperature. The idea behind the equilibrium model is that when under different temperature gradients, the inactive form of the enzyme that is added along with the active form, prevents a full inactivation of the active enzyme by equilibrium mechanics. It should be noted however that the difference between inactive and active forms of the enzyme must be understood. The equilibrium model describes the inactive form of the enzyme by purely centralizing its activity according to its active site. The active site of an enzyme is where substrates are capable of binding which then proceeds into a conformational change of the enzyme to then further proceed with the intended biochemical reaction. The inactive enzyme in this model is described as a mere folding change in comparison to the active form of the enzyme. This is not to be confused with an enzyme that has been denatured. An enzyme that is denatured is one that is completely changed, active site and all, to the point where the enzyme can not function at all, in other words, it is an irreversible enzyme conformational change. In the case of the equilibrium model, the mechanic of this model works simply because of the reversibility of the inactive enzyme form. The inactive form of the enzyme can reverse back to its active form which is in complete correspondence of the whole idea behind the equilibrium mechanics of the proposed model.
Berg, Jeremy "Biochemistry", Chapter 7 Hemoglobin: Portrait of a Protein in action. 193-194. sixth edition. Freeman and Company, 2007. http://hrsbstaff.ednet.ns.ca/sdosman/Higher%20level%20BIO/enzymenotes3.6.htm
Roy M. Daniel, Michael J. Danson, A new understanding of how temperature affects the catalytic activity of enzymes, Trends in Biochemical Sciences, Volume 35, Issue 10, October 2010, Pages 584-591, ISSN 0968-0004, 10.1016/j.tibs.2010.05.001.