Experiment #3: Enzyme Kinetics NOSOTROS, Ro-Ann S. ROGACION, Diara Jossiean M. Group #6, Chem 40.

1, WEJ2, Sir Kevin Sison January 22, 2014 I. Abstract Enzymes are protein catalysts that facilitate in the reactions of a system by increasing the rate of reaction and lowering the energy needed for it to proceed (activation energy) without being changed or consumed in the process. Most biological processes cannot take place in conducive rates without the help of enzymes. The level of pH, temperature, substrate concentration, time of reaction and the presence of inhibitors are some of the factors that can affect enzyme activity. For this experiment, a starch standard curve was constructed and the effect of reaction time, temperature and pH in solutions containing starch and salivary amylase were recorded as well. The resulting samples underwent spectrophotometric analysis to obtain their respective absorbance values so as to determine the optimal conditions for effective enzymatic activitythe lower the absorbance value, the more effective the enzymatic activity is, and the condition can therefore be said to be at the optimum level. It was found that effective activity of enzymes occurs at a longer incubation period, at around 40 C and at around pH 7.0, although a not all raw experimental data values give the theoretical result. Areas for the discrepancies appeared due to some experimental errors. II. Keywords absorbance, dissociation constant, maximum velocity, spectrophotometer, substrate III. Introduction = Biological systems of living organisms + [] are dependent on chemical reactions. However, these reactions cannot stand on their own, i.e. (where Vo is the initial reaction velocity, Vmax is they would proceed at extremely slow rates. the maximal velocity, Km is the constant and [S] Reactants, or substrates, are selectively is the substrate concentration), describes how channeled into more favorable pathways by initial rate of reaction is directly proportional to mechanisms of enzymatic reactions. Enzymes substrate concentration, and how the overall rate are biological catalysts that lower the needed of reaction varies depending on said activation energy for reactions to occur at rates concentration. The Lineweaver-Burke Plot with that are suitable for systems to carry out. the equation, Enzyme kinetics is the study of the rate at which an enzyme works. Enzymatic reactions occur at various rates based on different factors that may affect enzyme activity. For this experiment, different factors that affect enzyme activity, such as reaction or incubation period and changes in temperature and pH were applied. Observation and analysis of enzymatic reactions also supply values for the optimal environment where the enzymes work most favorably. L. Michaelis and M.L. Menten devised a model that explains the characteristics of enzyme-catalyzed reactions. In this model, it is assumed that the substrate concentration is much higher than that of the enzyme and that there is only one substrate present for each enzyme. The Michaelis-Menten Equation, 1 1 = + creates a straight line that can be used to calculate the values of Km and Vmax. The biological catalyst used and analyzed for this particular experiment is the enzyme, amylase. It is a digestive enzyme mainly found in the saliva of many organisms so as to break down and cleave large polysaccharides such as starch, particularly catalyzing the hydrolysis of -1,4-glycosidic linkages of these polysaccharides to yield maltose, D-glucose and oligosaccharides. Salivary glands and the pancreas primarily produce amylase. The major form of amylase that is found in humans and some mammals is called -amylase.

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At the end of this experiment, students are expected to be able to understand and explain the fundamental concepts of enzyme kinetics through the catalyzing activity of amylase on starch. IV. Experimental A. Preparation of Starch Standard Curve Using the table scheme below, starch solutions of varying concentrations were prepared in seven 10 mL test tubes. Volume 0.1% Volume Volume I2 Test starch dH2O solution Tube # solution (mL) (L) (mL) 1 (blank) 0.00 3.00 20.0 2 0.50 2.50 20.0 3 1.00 2.00 20.0 4 1.50 1.50 20.0 5 2.00 1.00 20.0 6 2.50 0.50 20.0 7 3.00 0.00 20.0 Freshly prepared I2 in KI solution (0.3% w/v I2 and 1.5% w/v KI) was added to the starch solutions from each of these 2.0 mL was diluted to 10.0 mL and immediately placed in cuvettes to measure the absorbance at 620 nm of the I2starch complex using an UV/Vis spectrophotometer. The standard curve was plotted using starch concentration vs. absorbance. B. Reaction of Amylase in Human Saliva Ten (10.0) mL of human saliva was diluted to 100.0 mL, taking 1.0 mL of the enzyme solution to mix with 9.0 mL of 0.1% starch solution in five separate 20 mL test tubes. The mixtures in their test tubes were incubated at room temperature for, respectively, 0, 3, 5, 7, and 10 minutes. Starch hydrolysis was then terminated by immersing the test tubes in boiling water for 3 minutes and 2.0 mL of distilled H2O and 1 drop of I2 solution were added after cooling. After dilution of 2.0 mL of the mixtures to 5.0 mL, the absorbance of each was measured at 620 nm. Absorbance readings were converted to starch concentration using the standard curve and concentration was graphed versus incubation time. Average reaction velocity was then computed by dividing change

in concentration by change in time per interval. The reciprocal of starch concentration, 1/[S], versus that of velocity, 1/[v] were graphed and KM and Vmax were calculated. C. Effect of Temperature on Enzyme Activity One (1.0) mL 0.1% starch solution, 3.5 mL 0.1 M phosphate buffer pH 6.7 and 0.50 mL 0.9% NaCl solution were mixed in four separate 10 mL test tubes. Each test tube was respectively pre-incubated for 5 minutes in water baths with temperatures 10C, 40C, 60C, and 80C. Five (5) drops of enzyme solution were added without removing the test tubes from the water bath and additional incubation for 10 minutes was done. Starch hydrolysis was terminated by immersion in boiling water for 3 minutes and the mixtures were cooled prior to taking 1.0 mL aliquot to add 2.0 mL of distilled H2O and 1 drop of I2 solution. Absorbance was measured at 620 nm and concentration converted from absorbance readings through the use of the standard curve was graphed versus temperature. D. Effect of pH on Enzyme Activity In five separate 10 mL test tubes, 1.0 mL 0.1% starch solution and 0.50 mL 0.9% NaCl solution were mixed. Varying pH (2.0, 4.0, 6.7, 7.4, 9.0) of 3.5 mL of 1.0 M phosphate buffer and 5 drops of enzyme solution were respectively added to each test tube which were then incubated at room temperature for 10 minutes. Starch hydrolysis was terminated through immersion in boiling water for 3 minutes and the test tubes were cooled before taking 1.0 mL aliquot and adding 2.0 mL distilled H2O and 1 drop of I2 solution. Starch concentration converted from absorbance readings at 620 nm were graphed versus pH.

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V. Results A. Standard Curve of Starch Concentration Table 1. Absorbance of Mixtures with Different Starch Concentration Starch Test tube Absorbance, Concentration (% # 620 w/v) 1 0.0000 0.000 2 3 4 5 6 7 1.2 1
Absorbance

(Mstarch (%w/v) ) Vstarch soln = (Mfinal ,% Mfinal ,% Mfinal ,%

v vstarch v vstarch

v vstarch

)(Vtotal )

0.1% 0.5 mL 15.1mL

= 3.31x103 %

0.0331 0.0662 0.0993 0.1324 0.1655 0.1986

0.543 0.636 0.563 0.661 0.844 1.093

Table 2. Enzymatic Reaction of Amylase in Human Saliva

Time (min) 0 3 5 7 10 Abs., 620 0.497 0.203 0.122 0.194 0.123 Starch Conc. (% w/v) 0.1179 0.0482 0.0290 0.0460 0.0292 v [] 0 0.0233 0.0096 0.0085 0.0056 1/v 0 43.000 104.049 117.056 178.056 1/[S] 8.479 20.759 34.541 21.722 34.260

0.8 0.6 0.4 0.2 0

0.1

0.2

0.3

Starch Concentration (% w/v)

Concentration (%w/v)

y = 4.214x + 0.201 R = 0.814

0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000 0

y = -0.007x + 0.092 R = 0.637

5
Time (min)

10

15

Figure 1. Standard Curve of Absorbance of Mixtures with Varying Starch Concentration r2 = 0.814 slope = 4.214 y-intercept = 0.201 Equation of the line: y = 4.214x + 0.201 KM= 20.965 Vmax = 4.975 Sample computations for Starch Concentration: Vdilution Vtotal = starch solution + dH2 O + I2 Valiquot Vtotal = 0.5 mL + 2.5 mL 1 mL + 20L 1 x 103 L 10 mL 2 mL

Figure 2. Absorbance of Amylase in Different Time Intervals 40 30 1/[S] 20 10 0 0 50 y = 0.133x + 12.12 R = 0.718 100 1/[V] 150 200

Vtotal = 15.1mL

Figure 3. Plot of 1/[S] versus 1/[v] of Amylase Reaction in Different Time Intervals

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r = 0.718 slope = 1.33 y-intercept = 12.12 Equation of the Line: y = 1.33x + 12.12 KM = 0.0592 Vmax = 0.0825 B. Effect of Temperature Table 3. Absorbance of Starch Solutions Under Constantly Increasing Temperatures
Test Tube Temp. (C) Starch Conc. (% w/v) Absorban ce ( 620)

3 2.5 2 1.5 1 0.5 0 0 5

pH

Absorbance

10

Figure 4. Experiment Graph of Varying pH vs. Absorbance of the Starch Solutions VI. Discussion A. Standard Curve of Starch Concentration The relationship between two quantities is represented by a standard curve. This curve is used to determine the value of unknown quantities from those that are more easily measured. In this experiment, absorbance was plotted against starch concentration (in %w/v) such that the slope of the best fit line of the curve produced may be used to compute for starch concentrations in the succeeding measures of factors that can affect enzymatic activity. This was done by dividing the absorbance values measured by the slope of the standard curve. It must be noted that the non-linearity of the linear regression of the curve may have been due to errors in that one absorbance value, particularly of test tube number 4, did not follow the theoretical linear increase of absorbance. The positive slope, however, indicates positive KM, and hence demonstrates the theoretically accepted relationship between substrate concentration and absorbance. B. Reaction of Amylase in Human Saliva Amylase is the enzyme found in human saliva and is responsible for the breakdown of starch into its monomers, glucose. These monomers occur in polymers of two types. Amylose occur as long, unbranched chains of Dglucose residues held together by (14) linkages and can vary in molecular weight. Amylopectin, on the other hand, is highly branched due to glycosidic linkages join glucose residues as (14) linkages and branch points, (16) linkages. Starch, due to its structure, is

1 2 3 4 1 0.8 0.6 0.4 0.2 0

10 40 60 80

0.027 0.115 0.110 0.181

0.115 0.486 0.463 0.766

Absorbance

50
Temperature ( C)

100

Figure 3. Experiment Graph of Various Temperatures in degrees Celcius vs. Absorbance of the Starch Solutions C. Effect of pH Table 4. Absorbance of Starch Solutions Under Varying pH
Test Tube pH Starch Conc. (% w/v) Absorban ce ( 595)

1 2 3 4 5

2.0 4.0 6.7 7.4 9.0

0.601 0.599 0.570 0.476 0.546

2.533 2.524 2.401 2.005 2.301

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able to aggregate iodine atoms which usually induce yellow or brown coloration when randomly distributed in the system. This ability of starch causes blue-black pigmentation of samples containing the polymer. When starch breaks down into glucose, these monomers no longer have the capacity to hold together iodine atoms and thus the yellow or brown coloration is observed. Since amylase breaks down starch to glucose, a positive indication of enzymatic action is the decrease of starch concentration as time interval increases, which indicates increase in the concentration of glucose in the sample. There will come a time, however, that all of the amylase molecules have substrate attached to them and hence will not be able to act on other starch molecules. This dependence of enzymatic activity (in terms of velocity) on substrate concentration is demonstrated in the MichaelisMenten plot.

1 1 1 = + []

Figure 5. Lineweaver-Burk Plot

(Retrieved from http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/E/EnzymeKinetics.html)

In the equation, the reciprocal of velocity the y value,

1

is

the slope, [ ] the x value, and

the y-intercept. This double reciprocal

equation proves useful and more convenient in that Vmax can be easily approximated in the plot. C. Effect of Incubation Time As illustrated in Figure 2, absorbance varies directly with time, which is demonstrative of substrate concentration varying inversely with time, indicating an increase in the hydrolysis products, which, in this case was glucose. Using the standard curve, the values of starch concentration at different time intervals were determined. The individual absorbance value of each setup was divided by the value of the slope of the standard curve and plotted against the reciprocal of velocity. This resulted to a linear graph with a positive slope, indicating direct variation of rate of reaction and substrate concentration. As in the standard curve, non-linearity of the plots may be attributed to one particular measure of absorbance that did not follow the theoretical relationships. This may also be caused by errors in the part of the person performing the experiment.

Figure 4. Michaelis-Menten Plot

(Retrieved from plant_physiology/enzymekinetics.shtml) http://plantphys.info/

The maximum velocity, Vmax, is the rate of the reaction at which all of the enzyme molecules are acting on substrate molecules and hence the reaction can only proceed below this limit even after the substrate concentration is increased. The dissociation constant, KM, can be plotted as the value of substrate concentration to which Vmax corresponds to. A more convenient equation for plotting enzymatic activity was devised in the slopeintercept form of a linear equation, called the Lineweaver-Burk equation.

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D. Effect of Temperature The effect of temperature on enzyme activity can well be demonstrated by a standard graph of the enzymes reaction velocity plotted against increasing temperature.

However, contrary to the theoretical graph, an increase in enzyme activity was seen at 80 C.This may be in part, an experimental discrepancy influenced by the students. 10 C refered to the part of the graph before the peak while 60along with 80 C should have exhibited abrupt decline beyond the optimum temperature of 40 C. E. Effect of pH Enzymes also have an appropriate working environment under a certainpH. Effect of pH on the enzyme activity shows a symmetrical graph thathighlights the peak as the optimum pH.

Figure 5. Standard Relationship between Temperature and Enzymatic Reaction Velocity

(Retrieved from content/uploads/2012/12/TEMP.png) http://www.namrata.co/wp-

As illustrated, the peak of a graph showing the effect of temperature on enzyme activity corresponds to the optimum temperature. Before the plot reaches its peak, it can be deduced that the rate of enzyme activity is proportional to the temperature; that is, the high the temperature, the faster the enzyme acts upon its substrate. In addition, the gradual increase in temperature ensures that activation energy can be achieved. The increase in reaction velocity of the enzyme is due to the fact that the number of molecules with enough energy to exceed the energy barrier has increased and the formation of products eventually occurs. However, as the temperature constantly continues in increasing, the increasing heat absorbed by the system leads to the deformation of the enzymes conformation and consequential denaturation This renders the enzyme no longer functional. The negative slope exhibited by the graph after which the optimum temperature has been achieved indicates enzyme denaturation. In the conducted experiment, the results showed an abrupt decline of the graph after the enzyme activity reached 40 C. This is because at thisparticular temperature, there is minimal to no starch present in the solution; the enzyme can easily break down the starch particles.

Figure 6. Standard Relationship between pH and Enzymatic Reaction Velocity

(Retrieved from http://wizznotes.com/wpcontent/uploads/2010/12/image0131.png)

Principally, the physiological pH between pH 6.7 and 7.4 are also considered as optimal pH for most enzymes, although not all enzymes fall under this pH range; the pH wherein maximum enzyme activity is reached varies for + every enzyme. This is a reflection of the H concentrations at which an enzyme is fully functional. For example, pepsin, which is found in the stomach, has a pH of 2 while trypsin,which is found in the intestine, has a pH of 8. Changes in pH or the concentration of + H may alter the ionic interfaces among the charged particles on the active site of the enzyme and may then affect the reaction velocity in many ways. Catalytic processes typically require specific chemical groups for the enzyme-substrate complex. In addition, enzymes work when the net charge of its

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constituent residues is zero (isoelectric point). Protonation of the carboxylate group is due to an acidic environment, which in turn may affect interaction with adjacent basic amino acids. At alkaline pH on the other hand, the amino group is deprotonated and this causes the reaction rate to decline. The experimental graph did not quite show a symmetrical figure. It can also be viewed that the solution with pH 6.7, which is a pH within the optimum range, exhibited a remarkable decline in absorbance. Theoretically, the solution with pH 7.4 should constitute the peak of the graph since it is closest to the optimum and physiological pH. Areas of error that likely have happened were from the lapse of time in between mixing the substances and measuring the absorbance, and possible contamination of the buffer solutions. VII. Answers to Guide Questions 1. What are the optimum conditions for the action of salivary amylases? Salivary amylases have optimum pH of 6.7 to 7.0 and optimum temperature around 37C, the normal body temperature. 2. Does salivary amylase continue to act in the intestine? Why or why not? Salivary amylase cannot continue to act in the intestine due to the basic environment (around pH 8.0) that is beyond optimum of the enzyme. 3. Name the substrate and the end product of the salivary amylase activity. What specific structural properties must the substrate have to be recognized and acted upon by salivary amylase? Starch is the substrate and glucose is the end product of salivary amylase activity. Starch has a specific type of glucoseglucose bond that only amylase recognizes and is different from the glucose-glucose bonds of other polymers such as cellulose. 4. An alternative assay for enzyme activity involves the DNS reaction. What is the basis for this reaction? Describe the DNS assay.

This method tests for the presence of reducing sugars bearing a free carbonyl group (C=O), the polymers of which are acted upon by -amylase. This employs the use of 3,5-dinitrosalicylic acid (DNS) which is reduced to 3-amino,5-nitrosalicylic (ANS) acid under alkaline conditions, which also involved the oxidation of the aldehyde or ketone group of the monomer to a carboxyl group. ANS absorbs light at 540nm and thus reflects a characteristic color. Side reactions dependent on the nature of the reducing sugar compete for the availability of DNS and therefore will reflect unique intensities of color. 5. At times, very low enzyme activity is observed. What could be the possible causes of low activity? Low activity of enzymes may be due to lower concentration of substrates or unfavorable conditions such as beyond optimum pH levels or higher body temperature. 6. How do inhibitors affect enzyme activity? Give examples. Inhibitors are any substance that counteracts with the rate of enzyme-catalyzed reactions. Different types of inhibition accounts for different inhibitory processes. Reversible inhibitors may form bonds with the enzyme, and may be diluted to regain enzyme activity. However, irreversible inhibitors cannot regain activity following dilution. Some inhibitors are specific to their enzymes, such as venom and insecticides that inhibit the activity of acetylcholinesterase by irreversibly binding at its catalytic site, thereby increasing acetylcholine's duration of activity, and the vasodilating drugs, catopril and enalapril, which contain an inhibitor that blocks the cleaving enzyme, angiotensin I, responsible for vasoconstriction, thus maintaining low blood pressure. To add to this, antibiotics such as penicillin and amoxicillin can also provide an irreversibly inhibiting action on bacterial enzymes. VIII. Conclusion and Recommendations Proteins, given their biological functions, are affected by different factors that can limit their activity. One particular class of proteins

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vital to living organisms consists of the enzymes, also known as biological catalysts, which assist in the metabolic processes the organism has to undergo in order to continue living. Enzymes, like other proteins, can be limited by temperature and pH, for which each enzyme has a characteristic optimum level that, when surpassed, can lead to denaturation and hence loss of function. In the study of enzyme kinetics two of the most convenient devices of demonstrating substrate-enzyme relationships are the Michaelis-Menten and Lineweaver-Burk equations. Rate of reaction was found to be not entirely dependent on substrate concentration but that they can act only up to a certain concentration when all enzyme molecules are attached and acting on substrate molecules and will then work in a rate never surpassing the maximum velocity, Vmax, even when concentration is increased. The rate of enzymatic reaction can be followed through monitoring and measuring the rate of products appearance or disappearance of substrates. Experiments on enzyme kinetics involve more manipulation of quantitative variables and hence accurate and precise measurements of reagents and substances used and keen following of successive steps in experimentation must be carried out. Equipment must also be used, set up, and cleaned correctly to avoid errors in the results. It is recommended that the manual procedures be revised in that the dilution of aliquots of mixtures prior to measuring the absorbance be not carried out anymore since this only resulted to almost clear solutions that the spectrophotometer might not have been able to correctly measure absorbance. IX. References

Maryland. Retrieved from http://www.eng.umd.edu/~nsw/ench485/ lab4a.htm Nelson, D. L. and Cox, M. M. (2008). Principles of Biochemistry. (5th ed.). New York, NY: W.H. Freeman and Company. Champe, P. C. and Harvey R. A. (1994). Lippincotts Illustrated Reviews: Biochemistry. (2nd ed.). Philadelphia, PA: J. B. Lippincott Company. Standard Curve. Retrieved January 21, 2014 http://biology.kenyon.edu/courses/biol09 /standard%20curve/intro.htm Stryer, L. Tymoczko, J. Berg, J. (2007). Biochemistry. (6th ed.). New York, NY: W.H. Freeman and Company.

I hereby certify that I have given substantial contribution to this report.

________________________ Ro-Ann S. Nosotros

________________________ Diara Jossiean M. Rogacion

Amylase: Exploring digestion and evolution through a molecular machine. SEP Lessons. Retrieved January 21, 2014 from http://seplessons.ucsf.edu/node/2443 Laboratory Manual in Biochemistry. (2013). Department of Physical Sciences and Mathematics, College of Arts and Sciences, University of the Philippines Manila. Sun Wang, N. Glucose Assay Dinitrosalicylic Colorimetric Method. University of

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