Lineweaver-Burk plots rates

Lineweaver-Burk Plot (Rate of reaction m. s/kmol)... 

The three reversible mechanisms for enzyme inhibition are distinguished by observing how changing the inhibitor s concentration affects the relationship between the rate of reaction and the concentration of substrate. As shown in figure 13.13, when kinetic data are displayed as a Lineweaver-Burk plot, it is possible to determine which mechanism is in effect. 

Lineweaver-Burk plot Method of analyzing kinetic data (growth rates of enzyme catalyzed reactions) in linear form using a double reciprocal plot of rate versus substrate concentration. 

FIGURE 14.18 Single-displacement bisubstrate mechanism. Double-reciprocal plots of the rates observed with different fixed concentrations of one substrate (B here) are graphed versus a series of concentrations of A. Note that, in these Lineweaver-Burk plots for singledisplacement bisubstrate mechanisms, the lines intersect to the left of the 1/v axis. 

In our previous work [63], we studied the hydrolysis kinetics of lipase from Mucor javanicus in a modified Lewis cell (Fig. 4). Initial hydrolysis reaction rates (uri) were measured in the presence of lipase in the aqueous phase (borate buffer). Initial substrate (trilinolein) concentration (TLj) in the organic phase (octane) was between 0.05 and 8 mM. The presence of the interface with octane enhances hydrolysis [37]. Lineweaver-Burk plots of the kinetics curve (1/Uj.] = f( /TL)) gave straight lines, demonstrating that the hydrolysis reaction shows the expected kinetic behavior (Michaelis-Menten). Excess substrate results in reaction inhibition. Apparent parameters of the Michaelis equation were determined from the curve l/urj = f /TL) and substrate inhibition was determined from the curve 1/Uj.] =f(TL) ... 

The enzymatic activities of intercalated GOx-AM P layered nanocomposites at various pH values and temperatures were compared with the native enzyme in aqueous solution. In both cases, characteristic linear plots consistent with Michalis-Menton kinetics were obtained. The Lineweaver-Burk plots indicated that the reaction rates (Vmax) for free and intercalated GOx (3.3 and 4.0 pM min 1 respectively), were comparable, suggesting that the turnover rate at substrate saturation was only marginally influenced by entrapment between the re-assembled organoclay sheets. However, the dissociation constant (Km) associated with the activity of the enzyme was higher for intercalated GOx (6.63 mM) compared to native GOx (2.94 mM), suggesting... 

Characteristically, within certain concentration limits, if a chemical is absorbed by passive diffusion, then the concentration of toxicant in the gut and the rate of absorption are linearly related. However, if absorption is mediated by active transport, the relationship between concentration and rate of absorption conforms to Michaelis-Menten kinetics and a Lineweaver-Burk plot (i.e., reciprocal of rate of absorption plotted against reciprocal of concentration), which graphs as a straight line. 

What is Lineweaver-Burk plot The intercept and slope of Lineweaver-Burk plot were obtained as 5.0 x 10 3 and 1.5 x 10 5, respectively. Calculate the value of km and maximum rate. 

For example, experimental data might reveal that a novel enzyme inhibitor causes a concentration-dependent increase in Km, with no effect on and with Lineweaver-Burk plots indicative of competitive inhibition. Flowever, even at very high inhibitor concentrations and very low substrate concentrations, it is observed that the degree of inhibition levels off when some 60% of activity still remains. Furthermore, it has been confirmed that only one enzyme is present, and all appropriate blank rates have been accounted for. It is clear that full competitive inhibition cannot account for such observations because complete inhibition can be attained at infinitely high concentrations of a full competitive inhibitor. Thus, it is likely that the inhibitor binds to the enzyme at an allosteric site. 

Full and partial noncompetitive inhibitory mechanisms, (a) Reaction scheme for full noncompetitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor prevents release of product, (b) Lineweaver-Burk plot for full noncompetitive inhibition reveals a common intercept with the 1/[S] axis and an increase in slope to infinity at infinitely high inhibitor concentrations. In this example, K =3 IulM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial noncompetitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor alters (reduces) the rate of release of product by a factor p. (e) Lineweaver-Burk plot for partial noncompetitive inhibition reveals a common intercept with the 1/[5] axis and an increase in slope to a finite value at infinitely high inhibitor concentrations. In this example, /Cj= 3 iulM and P = 0.5. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

In partial (hyperbohc) mixed inhibition (O Figure 4-12d), binding of inhibitor to a site distinct from the active site results in altered affinity of enzyme for substrate (by a factor, ot) as well as a change (by a factor, /i) in the rate at which product can be released from ESI. The effects of a partial mixed inhibitor on a Lineweaver-Burk plot depend upon the actual values, and on the relative values, of ot and fl. Once again, inhibitor plots can intersect the control plot above or below, but not on, the oeaxis, and to the left or to the right of, but not on, the y-axis. Because Vmax cannot be driven to zero, a maximum Lineweaver-Burk slope is reached at infinitely high inhibitor concentrations beyond which no further increase occurs. 

From a Lineweaver-Burk plot, the K and of this rate-limiting enzyme were calculated to be 4 X 10" M and 8 X 10 mmol/h, respectively. If the above experiment is repeated in the presence of simvastatin, which of the following values would be obtained ... 

We examined the effect of restricted conformation on the activation entropy by kinetic studies at various temperatures [34]. Three kinds of substrates were subjected to the reaction phenylmalonic acid as the standard compound, ortho-chlorophenylmalonic acid as a substrate with an electron-withdrawing group, and indane-l,l-dicarboxylic acid as a conformationally restricted compound. The initial rates of the enzymatic decarboxylation reaction of three compounds were measured at several substrate concentrations at 15 °C, 25 °C, and 35 °C. The kcat and values at each temperature were obtained by a Lineweaver-Burk plot,... 

Once the four anionic fractions were isolated (Bi, B2, Xi, X2), their activities were investigated using ferulic or / -fluoroferulic isopropylamine salts as substrates. Rates were plotted as a function of substrate concentration. The Lineweaver-Burk plots obtained (Fig. 4) were not always strictly linear as already reported in the case of ferulic acid and scolopetin oxidation (10,11)- An estimation was made of the apparent Km using the linear part of the plots and results were compared with those obtained for TMB. The values found in this case were in the same order of magnitude, about 0.5 X 10-3 to 1 x 10-3 M. In all extracts, / -fluoroferulic salt inhibited enzyme activity for concentrations higher than 0.25 X 10-2 M. 

The mixed-type inhibitors combine the effects of the competitive and noncompetitive inhibitors binding at the active center decreases the affinity of the enzyme towards the substrate molecule and also decreases the rate of transformation of the bound substrate. In their presence, the straight line plots intersect in the fourth quarter of the Lineweaver-Burk plot, according to equation ... 

Bimolecular reactions of two molecules, A and B, to give two products, P and Q, are catalyzed by many enzymes. For some enzymes the substrates A and B bind into the active site in an ordered sequence while for others, bindingmay be iii a random order. The scheme shown here is described as random Bi Bi in a classification introduced by Cleland. Eighteen rate constants, some second order and some first order, describe the reversible system. Determination of these kinetic parameters is often accomplished using a series of double reciprocal plots (Lineweaver-Burk plots), such as those at the right. 

Linear forms for rate equations. To obtain Km and Vmax from experimental rate data, Eq. 9-15 can be transformed by algebraic rearrangement into one of several linear forms. The popular double-reciprocal or Lineweaver-Burk plot of 1/ v against 1 / [S] (Fig. 9-3) is described by Eq. 9-20. The values of Km/ Vmax and 1 / Vmax can be evaluated from the slope and intercept, respectively, of this straight line plot. 

Second, an enzyme assay may be used to measure the kinetic properties of an enzyme such as Ku, Vmax, and inhibition characteristics. In this situation, different experimental conditions must be used. If Ku for a substrate is desired, the assay conditions must be such that the measured initial rate is first order in substrate. To determine Ku of a substrate, constant amounts of enzyme are incubated with varying amounts of substrate. A Lineweaver-Burk plot (1/v vs. 1/[S]) or direct linear plot may be used to determine Ku and V. If a reaction involves two or more substrates, each must be evalu-... 

The treatment of results will be described for L-dopa. The procedure for D-dopa is identical. Prepare a table of L-dopa concentration per assay (mmo-lar) vs. A/i/min. Convert all AA/mm units to /xmoles/min as described in part B. Prepare a Michaehs-Menten curve (/xmoles/min vs. [S]) as in Figure E5.1 and a Lineweaver-Burk plot (l//xmole/min vs. 1/[S]) as in Figure E5.2. Alternatively, you may wish to use the direct linear plot. Estimate Ku and Vmax from each graph. The intercept on the rate axis of the Lineweaver-Burk plot is equal to 1/V-. For example, if the line intersects the axis at 0.02,... 

To determine KM values, conduct rate measurements (v) with at least four different concentrations of any given substrate, [S], and then analyze the data by any suitable kinetic plot such as the Lineweaver-Burke plot or the direct-linear plot (refer to any standard textbook of biochemistry for more information). 

Flo. 5.14. Graphical representation of enzyme inhibition, (a) Eadie-Hofstee and (b) Lineweaver-Burk plots of different types of inhibition. The bold line indicates initial reaction rate in the absence of the inhibitor the lighter lines show initial rates in the... 

Figure 5. Lineweaver-Burk plots of amylose hydrolysis rates in the presence of the random copolymer at various temperatures. Temp (A) 85°, (A) 80°, (%) 75°, and (O) 70°C. [Catalyst] = 2.00 X W3N.

The inhibition effect of poly (vinyl alcohol) on the amylose hydrolysis was investigated. Figure 7 shows Lineweaver-Burk plots of the amylose hydrolysis rates catalyzed by the random copolymer in the presence of poly (vinyl alcohol). The reaction rate is found to decrease with increasing the concentration of poly (vinyl alcohol), and all of the straight lines obtained in the plots cross with each other at a point on the ordinate. This is a feature of the competitive inhibition in the enzymatic reactions. In the present reaction system, however, it is inferred to suggest that the copolymer and poly (vinyl alcohol) molecules competitively absorb the substrate molecules. The elementary reaction can be described in the most simplified form as in Equation 3 where Z, SI, and Kj[ are inhibitor, nonproductive complex, and inhibitor constant, respectively. Then the reaction rate is expressed with Equation 4. 

Figure 9 shows Lineweaver-Burk plots of dextrin hydrolysis rates in the presence of the block copolymer. Again, fairly good straight lines are obtained. Some other kinetical investigations also were made for the catalytic activity of the block copolymer, and similar tendencies of catalytic behavior were found compared with that of the random copolymer. 

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