Free Custom «Determination of Km» Essay Paper

Enzymatic reaction measurements are used in the determination of enzyme’s substrate affinities and their maximal reaction rates. Measurements taken are very important in the determination of microbial activities. Commercially available enzymes are commonly defined in terms of units and the unit value is specified for each individual enzyme. A unit of alkaline phosphatase is the amount of enzyme that can produce 1μmol of p-nitrophenol/minute. Alkaline Phosphatase are group of enzymes found in the liver, the bones, kidney and small linings in the intestines in humans. This enzymes work best at an alkaline pH making them inactive in the human blood.

Substrate concentration and Absorbance

V₀= V_max [S]/K_m+[S]    where [S] is the substrate concentration, V_max is the maximum enzyme velocity. K_m is the Michaelis-Menten constant while v is the enzyme velocity in unit of concentration of product per time and is normalized by enzyme concentration.

1/V₀ = 1/V_max + K_m/V_max*[S] which is the Line weaver-Burk equation.

Using the Beers Lambart law c=A/el              

1/V_max = 10, so V_max = 0.10

 

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−1/K_m = − 0.8, so K_m = 1.25 mM 

The accurate value of K_m is found at the intersection of the graph to the x axis where in this case the value is  -0.8 but the value at the intersection = -1/K_m thus K_m=1.25mM. This shows how much p-nitrophenol was generated by the enzyme per minute. K_m is the concentration that allows the enzyme to achieve ½ V_max. Enzymes with high K_m have a low affinity for their substrates and they normally require a larger concentration of substrate to achieve V_max.

When the concentration substrate is low there is a steep increase in the rate of reaction as the substrate concentration increases. The catalytic site of the enzyme has to wait for substrate to bind. This takes a longer time and the rate the product can be formed is limited to the concentration of substrate available. The substrate concentration increases as the enzyme becomes saturated with substrate. When the catalytic site becomes empty more substrate can be bound to undergo reaction. The rate of formation of product depends on the activity of the enzyme. This means that adding more substrate will not affect the rate of the reaction.

Every enzyme substrate complex has got its own unique K_m. The enzyme substrate complex is a function of pH and temperature. When we use p-nitrophenyl phosphate as a substrate instead of glucose-1-phosphate the value of Km will not be the same. Both reagents would react at different speeds in the presence of the enzyme alkaline phosphatase. P-nitrophenol was produced and it is yellow in color. This is done while the amount of the enzyme is kept constant and the substrate is increased. This causes the reaction velocity to increase until it reaches a maximum. After which increases in substrate concentration will not increase the velocity. The maximum velocity is reached when all the available enzymes are converted to the enzyme substrate complex thus a different K_m will be obtained. Another reason for change in the K_m may be due to change in the pH if the pH is extremely high or low then the activity will completely be lost. This will also affect the optimal stability of the enzyme.

Hyperphosphatasaemia is a syndrome that can be identified with the following properties: it has the heat stability characteristics of the bone isoenzyme but migrate on electrophoreses to a position slightly anodal to the liver isoenzyme alkaline. The mechanism leading to the abnormality is unknown but it is possible that the excessive sialylation results in loss of recognition by receptors that remove alkaline phosphatase from circulation, leading to very high plasma concentrations observed in this syndrome. Symptoms include; bone pain, nerve pressure, headaches and hearing loss and damage to cartilage of joints but it cannot diagnosed until a SAP is done and it checks raised alkaline phosphatase in the blood of the patient. Paget's disease is then confirmed with an X-ray of the bone or other tests, such as a bone scan. Bone scan helps to determine the extension of the disease for it provides visualization of the whole skeleton. The treatment of Paget's disease is focused on reducing its complications. No treatment is required if the disease causes no major symptoms and the SAP shows a small rise in serum alkaline phosphatase.