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Temperature y As the temperature rises, molecular motion and hence collisions between enzyme and substrate speed up.

But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective. y Temperature: enzymes work best at an optimum temperature. Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too. Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work pH y y The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected. as with temperature, enzymes have an optimum ph. If the ph changes much from the optimum, the chemical nature of the amino acids can change. This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured

enzymes are sensitive to their environmental conditions. Up to a point, the rate of the reaction will increase as a function of temperature because the substrates will collide more frequently with the enzyme active site. At extremes of pH or temperature, either high or low, the native structure of the enzyme will be compromised, and the molecule will become inactive (see Figure 3). Note that there is a sharp decrease in the temperature optimum for typical human enzymes at approximately 40 degrees Celsius (104 degrees Fahrenheit). At temperatures greater than 40 degrees Celsius, the enzyme degrades because the noncovalent interactions that stabilize the native conformation of the enzyme are disrupted. The enzyme in essence falls apart, and the active site is no longer able to function. In contrast, the optimal temperature for enzymes of the thermophilic bacteria (extremophiles) that live in hot springs is quite high at 70 degrees Celsius (158 degrees Fahrenheit), a temperature that would instantly scald skin. Enzymes also show a pH range at which they are most active (see Figure 3). The pH effect results because of critical amino acids at the active site of the enzyme that participate in substrate binding and catalysis. The ionic or electric charge on the active site amino acids can enhance and stabilize interactions with the substrate. In addition, the ability of the substrate and enzyme to donate or receive an H is affected by pH. The pH optimum differs for different enzymes. For example, pepsin is a digestive enzyme in the stomach, and its pH optimum is pH 2. In contrast, trypsin is a digestive enzyme that works in the small intestine where the environment is much less acidic . Its pH optimum is pH 8.

Concentration y the more of them available, the quicker the enzyme molecules collide and bind with them Enzyme concentration: at low enzyme concentration there is

great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor.

velocity increases as the concentration of the substrate is increased. Substrate concentration: at a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low. When more substrate molecules are added, more enzymesubstrate complexes can be formed. As there are more active sites, and the rate of reaction increases. Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed. Uv-vis spectro
y

Light from a light source will be passed through a test tube within which the reaction is occurring. The amount of light absorbed by the sample is directly proportional to the concentration of the light-absorbing chemical. After the light passes through this chemical, it is allowed to hit a photo-electric cell, which in-turn conducts electricity depending on the intensity of light hitting it. The magnitude of electricity conducted can be measured with an ammeter. Changes in the amount of light hitting the photocell allow measurement of changes in the concentration of dissolved chemicals.1

Another point worth noting is that as the reaction proceeds, bubbles would be produced within the test tube. The rate of bubble production is also proportional to the rate at which the reaction proceeds. This gives us another variable to work with. If the reaction is speeded up by increasing the concentrations of the reactions, more number of bubbles would be produced in a given time, and these bubbles would affect the amount of light allowed to hit the photo-electric cell. In the above reaction, the blue colour of the Cu(NO3)2 (aq) would be appropriate to measure. As the reaction proceeds, the intensity of the blue colour will increase. Plotting a graph of current versus time allows the slope of the graph to be found. This slope is nothing but the rate of production of Cu(NO3)2, or in other words, the rate of the reaction. y y Either he rate of appearance of product or the rate of disappearance of substrate is measured, often by following the change in absorbance using a spectrophotometer. http://docs.google.com/viewer?a=v&q=cache:QC5K0jsFpiAJ:www.life.illinois.edu/biochem/ 455/Lab%2520exercises/Photometry/spectrophotometry.pdf+filetype:pdf+%22spectrophot ometry%22+%22substrate%22+reaction+rate+enzyme&hl=en&gl=ph&pid=bl&srcid=ADGEE SgQ1JjPQW4MPwmsfFZ5OnksL25XMlTod7S_g4eFGlDEMp54Q8V2ryqR2DKV_CY5leBod2QU vkxVtSfQtJL_jGBDukYHo5MjMZ49odR0kRYTZFs1-j4t5UdmyZrRU9mnn63KhuA&sig=AHIEtbQwMDTlPpdSG62EP1qrZYSuB2lFCA An enzyme catalyses the conversion of one or several

substrates to one or several products. The rate of the catalysed reaction or the activity of the enzyme can be determined by measuring either the decrease in substrate concentration or the increase in product concentration as a function of the reaction time. When the substrate (S) and the product (P) di er in absorbance, the progress of an enzymatic reaction can be followed directly by monitoring the change in absorbance as a function of time. The absorbance changes are linearly related with the changes in concentration (via the Lambert Beer relation, see eqn [2]) and therefore the reaction rates (d[P]/dt or 2 d[S]/dt) can be calculated directly from the absorbance data when the absorption coe cients of the reacting species are known. NADH-linked enzyme reactions, such as those catalysed by the lactate, malate or alcohol dehydrogenases provide excellent examples for absorbance-based enzyme assays. The reduced nicotinamide ring in NADH shows an absorbance maximum near 340 nm (e340 5 6220 L mol 2 1 cm 2 1), which is lost upon oxidation to NAD 1 . Thus the activities of these dehydrogenases can be measured directly by following the decrease in A340 as a function of time. When no absorbance changes occur during the reaction of an enzyme with its natural substrate, often coloured derivatives can be synthesized with chromophoric reporter groups. A commonly used reporter is 4-nitrophenolate, which absorbs near 400 nm. This group can be esteri ed with acetic acid in 4-nitrophenyl acetate (to serve as a substrate for proteases), with phosphate (as a substrate for phosphatases) or with sugars (to probe amylases or glycosidases). In favourable cases a silent enzyme reaction with a colourless product can be coupled with another enzyme reaction that uses the product of the rst enzymatic reaction for a conversion that leads to a change in absorbance. Often such silent reactions are coupled to an NADH-consuming or NADH-producing step. As outlined above, changes in NADH concentration are easily followed by the strong change in absorbance at 340 nm. It is, of course, essential that in such coupled enzyme assays the rate of the indicator reaction is much higher than the rate of the primary reaction. This is usually achieved by using the indicator enzyme in a high concentration.