PH, enzyme activity

The activity of most enzymes depends on pH. The pH-enzyme activity profile of most enzymes delineates a bellshaped curve (Figure 6-3), exhibiting an optimal pH at which activity is maximal. This pH is usually the same as the pH of the fluid in which the enzyme functions. Thus, most enzymes have their highest activity between pH 6 and pH 8 (the pH of human blood is about 7.4). However, pepsin, which must function at the low pH of gastric juice, has maximal activity at about pH 2. 

The conditions of the reaction are important, particularly the concentration of cofactors, divalent ions, monovalent ions, temperature, and pH. Enzyme activity generally increases with temperature up to the point where thermal denaturation destroys its activity. Most enzymes have an optimum pH which commonly has a value between 6 and 8. The shape of the pH versus activity curve depends strongly on the enzyme. 

A pH-enzyme activity curve is shovm in Figure 9-1. Which of the following pairs of amino acids would be likely candidates as catalytic groups (See Primary Text, Table X-X for the pKa values of amino acid residues.)... 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

Potentiometry is another useful method for determining enzyme activity in cases where the reaction Hberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base requited is measured. An example of the use of this method is the determination of Hpase activity. The enzyme hydroly2es triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

In many situations, the actual molar amount of the enzyme is not known. However, its amount can be expressed in terms of the activity observed. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute. (Because enzymes are very sensitive to factors such as pH, temperature, and ionic strength, the conditions of assay must be specified.) Another definition for units of enzyme activity is the katal. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. Thus, one katal equals 6X10 international units. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

Bell-shaped activity versus pH profiles arise from two separate active-site ionizations, (a) Enzyme activity increases upon deprotonation of (b) Enzyme activity decreases upon deprotonation of A-H. (c) Enzyme activity is maximal in the pH range where one ionizable group is deprotonated (as B ) and the odier group is protonated (as A-H). 

Substrate (compound to be determined) Enzyme (activity to be determined) Products (sensed species) Buffer and optimum PH Indicating electrode Range... 

To establish the most advantageous conditions for production of L-phenylalanine from acetamidocinnamic add using two micro-organisms the following factors were investigated pH, amino donor and ratio of two enzyme activities. 

Under optimal conditions (pH = 8.0,67 g T1 L-aspartic add, 30°C, 1 1 ratio of enzyme activities) after addition of pyridoxal phosphate, 76 g l 1 L-phenylalanine could be produced within 72 hours (92% conversion). This illustrates how simple biochemical manipulation can increase productivity dramatically. 

Enzyme activity in organic solvents depends on parameters such as water activity, pH control, substrate-product solvation, enzyme form, and nature of the solvent. 

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

Figure 8-2. Effect of pH on enzyme activity. Consider, for example, a negatively charged enzyme (EH ) that binds a positively charged substrate (SH ). Shown is the proportion (%) of SH+ [ ] and of EH [///] as a function of pH. Only in the cross-hatched area do both the enzyme and the substrate bear an appropriate charge.

Available methods provide measurements of enzyme activity rather than of enzyme concentration. In order that the measured activity be proportional to enzyme concentration, the reaction conditions which include pH, temperature, initial substrate concentration, sample and total volume and reaction time must be held constant and be carefully controlled. 

The concentrations of substrate, coenzyme, buffer, activators, pH, etc., should provide maximum enzyme activities. 

Reagents. The measurement of enzyme activities requires rigid control of the analytical conditions, including accurate measurement of reagent and sample volumes, and careful control of temperature, pH and reagent stability. 

Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens 1AM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely... 

PL activity was determined by monitoring A236 for the formation of unsaturated products released from 0.1% polygalacturonic acid (P-1879, Sigma), dissolved in 0.1 M Tris/HCl buffer, pH 8,0 supplemented with 0.1 mM CaCb. One unit of PL is the enzyme activity liberating 1 pmol of unsaturated oligoglacturonides from pectate per min at 25°C. Activities are given in mU / ml extract per min. 

Effect of pH and temperature on the purified enzyme activity and stability The conditions of the enzyme activity and the stability was done followed Buranakarl, et al. (16). 

Effect of pH and temperature on the purified enzyme activity and stability... 

AE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Mannheim). The activity of AE was measured in 0.6% sugar beet pectin solubilised in 25 mM Na-succinate pH 6.2 and incubated with enzyme fraction in total 500 nl assay. The samples were incubated at 40°C and aliquots were examined after 0, 1, 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at lOO C for 5 min. Precipitated... 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

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