Secondary plot, enzyme kinetics

Second order reactions 458 Secondary kinetic isotope effect 592, 600 on fumarate hydratase 684 Secondary plots for kinetics of multisubstrate enzymes 465 Secondary structure 63... 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

Figure 9-6 Reciprocal plots used to analyze kinetics of two-substrate enzymes. (A) Plot of 1 / against 1 / [A] for a series of different concentrations of the second substrate B. (B) A secondary plot in which the intercepts from graph A are plotted against 1/ [B], (C) Secondary plot in which the slopes from graph A have been plotted against 1 / [B]. The figures have been drawn for the case that Kmp = 10 3 M, Kun, = 2 Km, and K B = KeqAKmB (Eq. 9-46) = KmJ 200 and [A] and [B] are in emits of moles per liter. Eadie-Hofstee plots of v / [A] vs vf at constant [B] can also be used as the primary plots. The student can easily convert Eq. 9-44 to the proper form analogous to Eq. 9-21.

Fig. 3. p-Glucosidase inhibition shown by Lineweaver-Burk plot (reproduced from [2]). Lineweaver-Burk plot of kinetic data from peak 2 cellobiase ((3-glucosidase) at several product inhibitor levels. This is an example of noncompetitive inhibition where the product is not only completing for binding in the active site but also binding to a secondary site on the enzyme that alters the enzyme catalytic ability... 

Fig. 6 Cyclic voltammetric analysis of the kinetics of an electrode coated with antigen-antibody immobilized monomolecular layer of redox enzyme with a one-electron reversible cosubstrate in the solution, (a) Cyclic voltammetry at saturation coverage (2.6 x 10 mol cm ) of glucose oxidase with 0.1 M glucose and 0.1 mM ferrocenemethanol in a pH 8 phosphate buffer (0.1 M ionic strength). The dotted and dashed lines represent the cyclic voltammogram (0.04 V sec ) in the absence and presence of glucose (0.1 M), respectively. The full line represents the catalytic contribution to the current,/ cat (see text), (b) Primary plots obtained under the same conditions with, from top to bottom, 0.01, 0.02, 0.05, and 0.1 M glucose, (c) Secondary plot derived from the intercepts of the primary plots in (b).

The results obtained from Lineweaver-Burk plots, are used for calculation of kinetic constants. The secondary plots of the slopes and intersects vs. activator concentrations are not linear (data not shown), but the reciprocal of the change in slope and intercept (Aslope and Ainiercept) that are determined by subtracting the values in the paesence of activator from that in its absence, are hnear. The intercepts of a plot 1/Aslope and 1/Airtercept 1/ [Al ] on 1/A axis, and intercepts of both plots on l/[ Al ] axis are used for calculating equilibrium constants Kms and Kma for dissociation of formed binary enzyme-activator (Al ) and ternary enzyme activator- substrate complexes (Figure 4). The calculated values for constants are (0.904 + 0.083) mM and (8.56 + 0.51) mM, respiectively. 

Anti-cancer drugs that are sulfamate esters, ROSO2NH2, appear to act by inhibition of sulfatases. Now, kinetic studies of the aminolysis of p-nitrophenyl sulfamate (109) by secondary alicyclic amines in MeCN at 310 K are reported that model the enzyme reaction. The Brpnsted-type plot was biphasic, the break point at w 18.2 more or less corresponding to the pKa of the ester (109) (17.8). The proposed mechanism (Scheme 30) involves a sequential double deprotonation of (109) leading first, via (110), to the sulfenamine (112) and at higher basic strength to (111) and thence to a novel anionic sulfenamine (113), the products in each case being an V, V-dialkylsulfamide (114) and p-nitrophenol.114... 

The rate equations for the several possible enzyme-substitution mechanisms all lack the last term of this equation. The kinetic coefficients in Eq. (2) can be estimated by primary, secondary, and tertiary plots 21). 

If a 2-substrate enzyme does not obey Eqn. 16 then this may show up either in the primary plots of e/v against 1/[substrate 1] or in the secondary replots of primary slopes and intercepts against 1/[substrate 2], or both. Detection of the non-linearity usually depends on using a wide range of substrate concentrations. Curvature is usually sufficiently gradual that it may be mistakenly dismissed as experimental error if only a narrow range is employed. As has been discussed earlier non-linear kinetics may be due to allosteric interaction or to alteration of bulk properties of the solvent at very high substrate concentrations. Non-linearity may. 

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