Experiments were put frontward to find how enzyme dynamicss maps under normal conditions and when the enzyme is capable to the effects of an inhibitor. Along with this process the enzyme is capable to a scope of pH ‘s to find the optimal rate and besides capable to a scope of temperatures individually to infer the optimum. A method used to find the optimal rate is known as spectrophotometry and this is of import. In basic footings it measures the substrates colour transition and shows how much visible radiation will go through through, therefore making so may or may non obey beer Ls jurisprudence. Using a standardization graph is indispensable to change over optical density to [ V ] rate of reaction per minute and is utile to assist build a michaelis-menten and lineweaver-burk graph to more accurately subtract the optimum. A similar set of graphs utilizing a curve and additive attack can infer how pH and temperature affect the enzyme ‘s activity. Some cardinal consequences related to the enzymes activity are Vmax ( an estimated speed rate upper limit ) and Km ( concentration of substrate that gives half the Vmax ) .Without inhibitor the Vmax = 66.6 micromoles/min this is determined by the overall speed and finding of the flattening of the curve at its extremum. Km = 0.0947mM which is determined after the Vmax is estimated. With Vmax= 116.7 and Km = 0.833. The enzyme topic to pH has a value of 0.00204 micromoles/min at pH 5. Temperature shows that 50 grades Celsius is an optimal disclosure 0.004420 micromoles/min. This consequences can be interpreted to demo that when an enzyme is the topic to alter It by and large follows a tendency picturing the enzymes optimum where more energy = higher [ V ] per/min and higher sums of substrate equate to a higher speed ( until the active sites are saturated ) .
General enzyme dynamicss
“ The usage of enzymes in the diagnosing of disease is one of the of import benefits derived from the intensive research in biochemistry since the 1940 ‘s. “ ( Worthington Biochem corp, 2012, hypertext transfer protocol: //www.worthington-biochem.com/introbiochem/default.html, accessed 15/11/12 ) Enzymes have provided the footing for the field of clinical chemical science. Catalysis is an of import factor in an enzymes map, it is an enzyme expeditiously used to increase a specific reaction e.g. – Hydrolases are enzymes that will catalyze a hydrolytic cleavage. Enzyme concentration corresponds with the correlativity of the substrate, which means more active sites are available to go concentrated ; this will increase the rate of the reaction. i.e. – Tocopherol + S & lt ; – & gt ; ES – & gt ; EP – & gt ; E + P. Substrate concentration may be increased up until the enzyme ‘s active sites are wholly saturated so that adding any longer substrate to the enzyme substrate composite will non increase or change the maximal rate of reaction ( Vmax ) . A measurement known as ( Km ) which is half the ( Vmax ) can show how tightly the substrate is bound, intending a low value of ( Km ) matching to a strong bond and frailty versa. This besides can show how fast the enzyme is catalyzing the reaction ; intending the lower the ( Km ) the faster the enzyme reaches its ( Vmax ) besides demoing that this happens in a lower concentration of substrate. ( B. Alberts et al. , 2007, Chapter 3: Proteins )
Datas from enzyme dynamicss can be interpreted by a Michaelis-menten graph ( shown Figure 1 ) . This graph demonstrates a typical conclusive enzyme reaction ; where a curve is shown stand foring reaction rate matching to substrate concentration. It outlines that a typical enzyme reaction will make a extremum and flatten out i.e.- Vmax. This illustration besides demonstrates the Km and by sing this point, can find the velocity of reaction rate. If the graph shows this patterned advance so it is obeying Michaelis-menten dynamicss.
Figure 1 ( CHEMWIKI. A.Nath University of Washington 2011 ) hypertext transfer protocol: //chemwiki.ucdavis.edu/ @ api/deki/files/13014/=mm1.gif
Michaelis-menten can be determined by utilizing similar estimated values ( substrate [ S ] ) and resubmitting them into the formulae V = Vmax x [ S ] /Km + [ S ] , This will demo a curve and dependent on the correlativity compared to the initial consequences, it could bespeak that the Vmax was over estimated ; i.e. – the theoretical curve will ab initio increase at a faster rate but will top out at a higher Vmax.
Using the same information a graph known as a Lineweaver-burk secret plan can be used to more accurately find a Vmax for the enzyme reaction rate. Since the Michaelis-menten graph does non stand for the informations accurately and the Vmax may hold been over estimated. A secret plan of a Lineweaver-burk graph is taken by the mutual
1/V = 1/ ( Vmax x [ S ] ) / ( Km + [ S ] ) this information can be plotted to demo the ( 1/Vmax ) which can be used to find the accurate Vmax instead than the estimated value. ( -1/Km ) shows a more accurate value for ( Km ) and so associating this to the enzyme reaction rate a higher ( Km ) means there is a lower affinity for the enzyme significance as antecedently discussed, there would be a strong bond in this instance.
Figure 2 ( Berg et al. , 2011 )
Inhibition is a procedure in which an ion or molecule will impact the activity of an enzyme. Inhibitors play a critical function because they are used in the organic structure for ordinance. E.g.- ” The ordinance of allosteric enzymes. ” ( Berg et al. , 2011 ) Figure 2 shows a diagram of a substrate binding to an enzyme active site ; this is expected of the enzyme under normal conditions. A competitory inhibitor will make as it suggests, it will try to acquire to the active site and bind before the substrate forestalling contact action. A competitory inhibitor can be reversed by increasing the ratio of substrate: inhibitor i.e.- higher concentration of substrate overrules the inhibitor to adhere to the enzyme making an enzyme – inhibitor composite. An uncompetitive inhibitor will merely adhere to the enzyme when it is in an enzyme-substrate composite this is because the binding site is merely available to the inhibitor in an ES complex – it is created when the substrate binds. A non-competitive inhibitor is where the inhibitor will adhere independently to the enzyme whether it is an ES composite or non. It will diminish the overall map of the enzyme instead than forestall the binding of the substrate, this is because of a conformational alteration to the active site forestalling it from adhering, and this is why increasing the concentration of the substrate will hold no consequence.
A Michaelis-menten secret plan can besides be used to demo that there is still an initial addition in reaction rate because non all the enzyme is inhibited ; bit by bit the enzyme becomes an EI or ES complex. On its ain the graph secret plan of an inhibitor can non demo basically how much it has affected the reaction rate. To demo this it is plotted along the secret plan of what would be expected if the substrate can efficaciously adhere.
Figure 3 is an illustration of what is expected when an repressive experiment on an enzyme is compared alongside the same experiment with the enzyme under normal conditions. The competitory inhibitor will make the same Vmax because the substrate can still adhere with the active site, while viing. The non-competitive inhibitor will hold a lower Vmax due to it adhering outside the active site ; as shown on the graph it is obvious how a non-competitive inhibitor can impact the Vmax and the Km. hypertext transfer protocol: //users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Michaelis_Menten2.gif
Figure 3: Enzyme dynamicss, hypertext transfer protocol: //users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html, 2011, accessed 12/11/12.
As explained before a michaelis-menten secret plan does non find accurately the Vmax and it could most likely have been overestimated for the inhibitory curve. A lineweaver-burk secret plan is used to demo how affectional the inhibitor has been upon the Vmax in comparing to normal conditions.
Figure 4 shows the lineweaver-burk secret plan of an inhibitor/s compared to the enzyme reaction under normal conditions ( shows as the ruddy line ) . As shown in figure 3 the non-competitive inhibitor has a lower Vmax and this graph besides shows more efficaciously that the active site is n’t affected by the inhibitor. This is shown by the ( Y ) intercept. hypertext transfer protocol: //users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/Lineweaver_Burk2.gif
Figure 4: Enzyme Dynamicss, hypertext transfer protocol: //users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html, 2011, accessed 12/11/12.
Temperature is an of import co-factor that can change the reactive province of an enzyme. It can expeditiously rush up the reaction rate up until the denaturation of the enzyme. Denaturation occurs when the temperature exceeds the enzymes optimal and hence the H, inter and intra-molecular bonds are no longer keeping the enzymes conformation together ( Average kinetic energy has disrupted the molecules in the conformation ) . This indefinitely means that a substrate can non adhere to the active site because it has lost its signifier due to the denaturation. ( RSC, hypertext transfer protocol: //www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm, n/a, accessed 13/11/12 )
Typically as the temperature raises the reaction rate increases up until a peak known as the optimal temperature at which the enzyme will work. This can be shown by a graph.
Figure 5: This curve represents the effectivity of temperature on an enzymes activity. Basically the temperature addition will give the enzyme more kinetic energy up until that energy is strong plenty to denature the conformation. Notice that the line does non stop at “ 0 ” this is because the reaction is taking place really easy beforehand at lower temperatures, intending less kinetic energy for the substrate and enzyme. Graph of enzyme activity poetries temperature
Figure 5: Enzymes, hypertext transfer protocol: //www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm, RSC, 2011, accessed 13/11/12
A much easier manner to see the optimum temperature for an enzyme ( i.e. – when it denatures ) is to plot Log10 of the rate alongside 1/T ( which is temperature in K ) . Arrhenius equation is used to plot this: K = A -Ea/RT.
Where K = Rate invariable, A = Pre-exponential factor ; stand foring figure of hits Log10 C ( where C = proportionality invariable ) Ea = Activation energy of rate of reaction, R = Gas Constant ( 8.31JK-1mol-1 ) T = Temperature ( K ) ( G.Sibert, hypertext transfer protocol: //www.files.chem.vt.edu/RVGS/ACT/notes/temp_effects.html, accessed 13/11/12 )
Figure 6 shows a additive manner utilizing the Arrhenius secret plan and can find the denaturation point more easy and accurately. The denaturation at optimal temperature is indicated by the anomalousness or isolated point. This graph is decidedly clearer to read and find. Cold temperature will decelerate down the enzyme activity by diminishing molecular gesture and kinetic energy. ( G E.Kaiser, 2001, hypertext transfer protocol: //student.ccbcmd.edu/~gkaiser/biotutorials/proteins/enzyme.html, accessed 13/11/12 )
Consequence of pH
The pH is a step of H ions, there for a high pH is high concentration of H+ and low pH is low concentration. An enzyme can be really delicate when topic to pH alteration. If the pH extends beyond the optimal the enzyme will denature. This can impact the ionization of amino acids which affects the conformation, taking to a alteration in the active site and map of the enzyme. These alterations may besides hold a great consequence on the weaker charges that keep the enzyme construction together ; it may besides change the substrate composite so that it is unable to adhere to the active site. All enzymes are alone to a map and some can work in pH degrees which others can non e.g. – in extremely acidic conditions such as the tummy, pepsin maps in the tummy and its optimal pH is really low because of these conditions. An enzyme can besides work in the complete opposite pH such as trypsin, which operates at a higher pH ( alkaline ) . Below is some illustrations, presuming the environment pH is 7.
Figure 7a is an illustration of a substrate binding to the active site of an enzyme. Normal conditions mean the Carboxyl ( COO- ) group can adhere to the amino ( NH3+ ) group making a substrate enzyme composite.
( a ) ( B ) ( degree Celsius ) hypertext transfer protocol: //www.chemguide.co.uk/organicprops/aminoacids/asiteph1.gif
Figure 7a, J.Clark ( a ) , hypertext transfer protocol: //www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html ( B ) , accessed 13/11/12 ( degree Celsius )
Figure 7b, is an illustration of what happens when the pH in the environment lowers, because this is an addition in H+ ions, the Carboxyl group will derive an H+ . This means that ionic bonds can non organize and the substrate will non adhere to the active site. Besides new bonds are formed in other places altering the conformation of the enzyme and its active site. Overall charge = Negative
( a ) ( B ) ( degree Celsius )
hypertext transfer protocol: //www.chemguide.co.uk/organicprops/aminoacids/asiteph2.gif
Figure 7b, ( a ) , ( B ) , ( degree Celsius )
Figure 7c, This is an illustration of when the pH increases so that the environment becomes alkaline, because this is a low H+ concentration it does n’t impact the carboxyl group, alternatively the amino group will lose a H+ ion efficaciously interrupting the active site bonding. Overall charge = Positive ( a ) ( B ) ( degree Celsius )
Figure 7c, ( a ) , ( B ) , ( degree Celsius ) hypertext transfer protocol: //www.chemguide.co.uk/organicprops/aminoacids/asiteph3.gif
PNP Calibration Graph
Sum of PNP ( Aµ/mol )
Absorbance ( 400nm )
Table 1 represents known concentrations of P-nitrophenol ( PNP ) , which is the merchandise formed after the enzyme is introduced. 0
Graph 1is plotted in conformity to the consequences of table 1, so the graph represents how the concentration can impact the optical density more clearly than reading it entirely. Simply the optical density represents how much visible radiation travels through the solution, in this the instance the solution has a dedicated sum of PNP, and as represented by the graph the higher the concentration of PNP the higher the optical density, i.e. – because less light can go through the PNP molecules.
Substrate ( PNPP ) Concentration ( millimeter ) [ S ]
Optical density of Sample @ 400nm
Optical density[ V ] per 15 min [ V ] per min ( I?moles/min )
n/2.6387= [ V ] p/15min[ V ] p/15min/15 = [ V ] p/min
Experiment A Effect of a substrate ( Without inhibitor )
This tabular array represents the informations collected from experiment A where optical density is relevant to find the rate of reaction. The first portion of table 2 indicates how the addition of substrate concentration correlatives to absorbance i.e. – Higher concentration is equal to an addition in optical density. The optical density collected from this experiment is used and converted into rate per minute or speed of the reaction. This is worked out by spliting the optical density by the Y intercept ( 2.6387 ) from the standardization graph and so spliting by 15 ( since the experiment was timed over a period of 15 proceedingss ) .
Michaelis-menten Graph ( chart 2 ) Consequence of a substrate ( without inhibitor ) Values from table 2.
Maximal Rate ( Vmax ) = 0.015 Aµmoles/min Km ( millimeter ) = A?Vmax = 0.17mM
The Vmax shows the point at which the active site ab initio becomes saturated ; this is an estimated value based on the secret plan of consequences therefore it is possible to overrate it. Km shows the affinity for the enzyme ; low Km = high affinity and high = low affinity.
Theoretical Valuess to find curve dependability[ S ]
Rate ( V )
Since the Vmax could hold been overestimated some theoretical values can substituted into the Michaelis-menten equation: Rate ( V ) = Vmax x [ S ] /Km + [ S ] i.e. – Rate ( V ) = 0.015 x [ S ] /0.17 + [ S ] . Using this to the values in table 3 will demo a curve, this curve is plotted ruddy on graph 2 ; the line can find whether the Vmax was overestimated or underestimated.
Lineweaver-burk – Consequence of a substrate ( without inhibitor )
1/ [ V ]
1/ [ S ]
( Table 4 ) This tabular array represents the reciprocal of the values in table 3. These values are used to plot a lineweaver graph which shows the rate of the reaction in a additive manner, doing it easier to find maximal rate. This is plotted on graph 3.33.33333333
Graph 3 is a lineweaver-burk secret plan and it represents how clearly the additive manner can demo the patterned advance of the rate of reaction. The smaller the figure the bigger the reciprocal is hence the higher values are smaller when plotted in a Michaelis-menten format. This graph can besides be used to find a more accurate Vmax and Km utilizing the reciprocal of the Michaelis-menten expression ;
i.e. – 1/V = 1/ ( Vmax x [ S ] / ( Km + [ S ] ) hence is in the format Y = maxwell + degree Celsius.
The Km can be more accurately determined by rearranging the expression so that Y is equal to 0, therefore:
0 = 8.636x + 83.728, therefore 0 – 83.728/8.636 so 1/9.69 = 0.103mM = Km the reciprocal of the Y intercept ( 83.728 ) is equal to 0.0119Aµmoles = Vmax
i.e. – means the initial Vmax was overestimated. Basically since both graphs are plotted with the same information, it is obvious that the Vmax ab initio was n’t right.
Consequence of a substrate ( With inhibitor )
Substrate PNPP ( millimeter )
( Table 5 ) This shows similar informations to ( table 2 ) but the rate of reaction is noticeable slower. This graph shows the optical density of visible radiation for the substrate, finding rate. Absorbance ( 400nm )
Optical density[ V ] per 15min
( Graph 4 ) shows clearly the difference between how an enzyme is affected by an inhibitor. A- Representing the merely substrate and enzyme, B stand foring the inhibited enzyme. Valuess determined by Michaelis-menten secret plan:
( With inhibitor ) Vmax = 0.0086 I?moles/min Km = 0.47mM ( low affinity )
( Without inhibitor ) Vmax = 0.015 I?moles/min Km = 0.17mM ( high affinity )[ V ] per min
Lineweaver-burk ( With Inhibitor )
1/ [ V ]
( Table 6 ) This represents the reciprocal of the rate of reaction and sum of substrate relevant to the inhibitor. ( Graph 4 ) This shows a more clear position of how an inhibitor affects enzyme reaction rate via a additive manner.
Without inhibitor: Vmax = 0.0119I?moles/min Km = 0.103mM
With inhibitor: Vmax = 0.0117I?moles/min Km = 0.833mM
1/ [ S ]
( Table 7 )
Temp ( grades C )
Absorbance ( 400nm )
( Abs [ 400nm ] )
( Abs [ 400nm ] )
Subtracted values ( 400nm )[ V ] /15min [ V ] /min
( Table 7 ) Represents the experimental tubings used and the effects of temperature on optical density by a curve. Graph 5 shows how the reaction rate increases up until where the enzyme denatures.
log ( V )
1/T is 1/ temperature and the temperature is measured in K therefore 1/k are the values in column ( 1/T ) i.e. – 1/278.2 = 0.00359K
Liner secret plan of temperature
( Table 8 )
Table 8 shows the Log 10 values of the rate of reaction [ V ] /min and the 1/t or temperature in K. Graph 6 shows a clearer position of the consequence of temperature on the enzyme utilizing a additive manner ; the anon. consequence is denaturation.
Effectss of pH on an enzyme
Abs ( 400nm )[ V ] /15min [ V ] /min
Table 9 shows the experimental values of the pH experiment, stand foring optical density matching to pH therefore incorporated into reaction rate. The experiment spanned over a period of 15minutes hence each sample is [ V ] /15 = [ V ] min. This information is represented on graph 7 where a curve is shown to make a extremum ; where at the optimal pH is for the enzyme activity rate. Estimated optimum of pH = 5.1
This is because p-nitrophenylphosphate ( PNPP ) is chromogenic ; in alkalic solutions it is colorless and alterations pigment in acidic solutions hence puting this ab initio into trial tubings with in scopes of pH ‘s of acidic to alkaline would alter the pigment, doing the experiment inaccurate because the measurings of optical density are taken from merchandise formed after the enzyme acid phosphatase is introduced. So the enzyme is left to incubate in the scope of pH ‘s because it is n’t chromogenic and acerb phosphatase, as it indicates maps good in acidic environments. The pH may get down to change the enzymes conformation as the solution reaches alkaline pH ‘s, due to the fact it starts to neutralize the acerb phosphatase.
Question2: The effects of pH on an enzyme are due to the addition in H+ when the pH is acidic or a lessening in H+ which is alkalic. Acidic amino acids have a carboxyl group in the side concatenation where alkaline amino acids have an amino group in their side concatenation. This means an addition or lessening in H+ ions can alter the province of the bonds, significance that the construction of the enzyme is altered. Besides an addition or lessening in protons can change the charge province of the substrate, forestalling it from adhering to the active site.
First an experiment took topographic point in which the enzyme undergoes its catalytic procedure in normal conditions to see and compare the consequences to farther probe and so it can be referred back to. Graph 2 is a Michealis-menten secret plan of the initial informations and it shows that the consequence of substrate concentration on the enzymes activity is determined by how efficient the active site is working. Therefore finally the active site will go concentrated and it will make a extremum. Graph 2 represents how the initial activity of the enzymes production accelerates at a crisp rate, and bit by bit extremums of to a level gradient analogue to the x axis. This shows that the enzymes active site is bit by bit going saturated, until there is a point at where the substrate concentration wo n’t impact the enzymes activity. The Vmax ( 0.015I?moles/min ) from this graph demonstrates the point at which the enzyme is saturated ; it may non be accurate due to estimation so a expression ( Rate = Vmax x [ S ] /Km + [ S ] ) can be used to plot some theoretical values to demo whether the Vmax was over estimated. This can intend that increasing the substrate concentration further in the initial experiment could demo a slow addition to a higher Vmax. It besides shows that the line follows the anticipations as so in the debut. The Km ( 0.17mM ) on graph 2 is interpreted by half of the Vmax read of at the x axis, therefore graph 2 shows a low Km, so the enzyme substrate composite is strongly bonded together and there is rapid catalytic acceleration. Graph 3 can be used to demo how the enzymes active site becomes saturated in a additive manner. In this instance little values go big and frailty versa, so the values for the experiment: Vmax = 0.0119Aµmoles/min and Km = 0.103mM, therefore the Vmax is how fast the substrate collides with the enzyme and binds to its active as demonstrated by a diagram ( figure 2 ) in the debut, utilizing a typical lock and cardinal method.
Second the same experiment was reproduced but with an inhibitor nowadays, similar information was recorded as experiment one, but the values have a noticeable alteration when the tabular arraies are compared. When the information is plotted on a lineweaver-burk ( graph 4 ) alongside the effects of the substrate entirely, graph 4 shows the additive relationship between the two gradients and shows that the inhibitor so does follow anticipation as stated in the debut. Along with this graph 4 is demoing that the inhibitor largely affects the active site because it is a competitory inhibitor, which explains why the Vmax ( 0.0117Aµmoles/min ) is closely related to when an inhibitor is non present, i.e. – the initial rate of reaction is predominately the same when compared but with an inhibitor present the gradient of the incline begins to flatten out before. The substrate can still adhere to the active site even with the inhibitor nowadays. The Km ( 0.833mM ) when compared with out an inhibitor ( 0.103mM ) nowadays shows a really low affinity so this means that the there is a weak bonding visual aspect in the substrate-enzyme composite, which is underlined by the fact that inhibitors prevent the right binding initial province of reaction.
The rudimentss of a correlativity correspond between [ S ] and [ V ] or the rate and this shows how the enzymes activity can match with a additive manner.
Another experiment expands the grounds of enzyme activity with a substrate, graph 5 expressions at how temperature and how an addition will impact the conformation of the active site and its binding with a substrate. Graph 5 shows that with general addition in temperature the enzymes-substrate complex binding becomes more efficient up to a extremum ; basically the addition in temperature besides increases the sum of hits between substrate and enzyme molecules. At the extremum on graph 5 which is at 49 grades Celsius shows where the enzyme has reached its optimal efficiency, this means the maximal sum of substrate is adhering with the active sites and potentially causing impregnation. Naturally after the optimal the enzyme starts to denature because the high temperature interruptions weak non-covalent forces such as H bonds that keep the conformation together for map, hence mean kinetic energy has disrupted the molecules in the conformation. This is shown by the steep incline after the extremum on graph 5, whereas a temperature addition corresponds to a lessening in the rate of production. The information for this experiment can be interpreted a batch better by plotting it in a additive manner which is represented by graph 6 utilizing log10 of rate of reaction plotted against 1/T ( 1/K ) . The ground for this secret plan is it shows where the denaturing of the enzyme takes topographic point harmonizing to temperature by the anomalous secret plan on the graph.
Temperature is n’t the lone factor, pH can alter the environment and change the conformation of the enzyme so it denatures. The step of pH is H+ ions and these can jump charges between the substrate and enzyme-complex, mentioning to the debut figure 7b and 7c represent how the amino and carboxyl groups are affected by the alteration in positive charge. This can demo how changing the substrate or/and enzyme active site can hold an consequence on the rate of binding. Since the enzyme is based to work in the kidney ‘s and liver ( acerb phosphatase ) it by and large functions better at mid-low pH ( 5 ) ( higher charge of H+ ions ) and graph 7 shows the correlativity of how the rate of merchandise produced corresponds with the addition in pH. After the optimal point graph 7 shows that the enzyme has started to be altered by the alteration in charges due to increase in pH ( lessening in H+ ions ) this can be explained by the illustration shown in figure 7b where the causality is possible loss of H ions from the carboxyl group therefore forestalling the substrate from adhering expeditiously or at all.
Enzymes and substrates dependant on their environment have the possible to change their activity between one another therefore, it concludes that enzyme-substrate composite is an of import procedure and these set of experiment show how infinitesimal alterations can act upon a co-factor of the binding activity. When compared with background informations the experiment has produced similar consequences reasoning that the consequences are accurate for representation of a co-factor ‘s consequence on substrate-enzyme composites. Transporting out the experiment more than one time with different and more refined precise measurings i.e. – utilizing a smaller scope of pH or temperature with denary values to find a more accurate consequence ; this will reason the overall experiment for the enzyme to be more dependable.