Substrate saturation curve

FIGURE 14.7 Substrate saturation curve for au euzyme-catalyzed reaction. The amount of enzyme is constant, and the velocity of the reaction is determined at various substrate concentrations. The reaction rate, v, as a function of [S] is described by a rectangular hyperbola. At very high [S], v= Fnax- That is, the velocity is limited only by conditions (temperature, pH, ionic strength) and by the amount of enzyme present becomes independent of [S]. Such a condition is termed zero-order kinetics. Under zero-order conditions, velocity is directly dependent on [enzyme]. The H9O molecule provides a rough guide to scale. The substrate is bound at the active site of the enzyme. 

FIGURE 15.11 Heterotropic allosteric effects A and I binding to R and T, respectively. The linked equilibria lead to changes in the relative amounts of R and T and, therefore, shifts in the substrate saturation curve. This behavior, depicted by the graph, defines an allosteric K system. The parameters of such a system are (1) S and A (or I) have different affinities for R and T and (2) A (or I) modifies the apparent for S by shifting the relative R versus T population. 

The converse situation applies in the presence of I, which binds only to T. T binding will lead to an increase in the population of T conformers, at the expense of Rq (Figure 15.11). The decline in [Rq] means that it is less likely for S (or A) to bind. Consequently, the presence of I increases the cooperativity (that is, the sigmoidicity) of the substrate saturation curve, as evidenced by the shift of this curve to the right (Figure 15.11). The presence of I raises the apparent value of L. 

In this form, Alhas the units of torr.) The relationship defined by Equation (A15.4) plots as a hyperbola. That is, the MbOg saturation curve resembles an enzyme substrate saturation curve. For myoglobin, a partial pressure of 1 torr for jbOg is sufficient for half-saturation (Figure A15.1). We can define as the partial pressure of Og at which 50% of the myoglobin molecules have a molecule of Og bound (that is, F= 0.5), then... 

In contrast to the kinetics of isosteric (normal) enzymes, allosteric enzymes such as ACTase have sigmoidal (S-shaped) substrate saturation curves (see p. 92). In allosteric systems, the enzyme s af nity to the substrate is not constant, but depends on the substrate concentration [A]. Instead of the Michaelis constant Km (see p. 92), the substrate concentration at half-maximal rate ([AJo.s) is given. The sigmoidal character of the curve is described by the Hill coef cient h. In isosteric systems, h = 1, and h increases with increasing sigmoid icity. 

Correction for endogenous substrate present in enzyme preparations is difficult. Measuring the endogenous rate (obtained by omitting the substrate involved) and subtracting this from the overall rate is generally incorrect (R5). Due to the nonlinearity of the substrate saturation curve, the endogenous rate will be more important in the control mixture than... 

Obviously, extrapolation procedures are impractical for routine determination of enzyme activities. When substrate saturation-curves conform to rectangular hyperbolas, reasonable concentrations of substrates should equal 10 to 20 times the respective Km values. As outlined above, application of this rule to assays of bilirubin UDP-glycosyltransferase activities is hampered by substrate inhibition and by occasional deviation from Michaelis-Menten kinetics. The best alternative in such cases may be to choose the concentrations at optimal enzyme activity. However, great care should be exercised in interpreting the results. When a bio-... 

The first product of nitrosyl transfer to nitrite in Eq. (2), E N203, contains N-N bonded N2O3 which is itself a well-known and powerful nitrosyl donor. It is reasonable to suppose therefore that nitrosyl transfer reactions with N- and O-nucleophiles could involve both E NO (or E HONO) and E N205. In addition, the involvement of a second molecule of nitrite for denitrification would require that the substrate saturation curve should be sigmoidal to reflect a term second-order in nitrite concentration. No such effect has been reported to our knowledge. The use of bimolecular substrate kinetics in dilute solutions to generate an intermediate subject to solvolysis seems metabolically unwise hut not impossible. 

Lineweaver-Burk analysis using the substrate saturation curves afforded the corresponding Michaelis-Menten kinetic parameters of the reaction V max=l-79 xIO- Ms , KM=21.5mM, kcat = 8.06x 10 s for 69, and Knax = 9.22x 10... 

For heterotropic allosteric enzymes, those whose modulators are metabolites other than the normal substrate, it is difficult to generalize about the shape of the substrate-saturation curve. An activator may cause the curve to become more nearly hyperbolic, with a decrease in Z0.5 but no change in Fmax, resulting in an increased reaction velocity at a fixed substrate concentration (V0 is higher for any value of [S] Fig. 6-29b, upper curve). 

Other heterotropic allosteric enzymes respond to an activator by an increase in Fmax with little change in if0i5 (Fig. 6-29c). A negative modulator (an inhibitor) may produce a more sigmoid substrate-saturation curve,... 

Substrate B does not change the regioselectivity of substrate A The regioselectivity of the enzyme is determined by the interactions between the substrate and the active site. Since the substrate saturation curve is defined by the Km of the enzyme, regioselectivity cannot be a function of substrate or inhibitor concentration [I],... 

In the presence of activator, pyruvate, the substrate saturation curves of the R. ruhrum ADP-Glc PPase are hyperbolic at low temperatures. Using kinetic studies its reaction mechanism was studied. The product inhibition patterns eliminated all known sequential mechanisms except the ordered BiBi or Theorell—Chance mechanisms. Small intercept effects suggested the existence of significant concentrations of central transis-tory complexes. Kinetic constants obtained in the study also favored the ordered BiBi mechanism. In addition studies using ATP-[ P]-pyrophosphate isotope exchange at equilibrium supported a sequential-ordered mechanism, which indicated that ATP is the first substrate to bind and that ADP-Glc is the last product to... 

Decrease in cooperativity of substrate saturation curve. Effector A lowers the apparent value of L. 

Thus, I inhibits association of S and A with R by lowering Rq level. I increases cooperativity of substrate saturation curve. I raises the apparent value of L. 

The kinetics of the transfer of the 2 -desoxyribosyI group from 2 -desoxythymidine to adenine is very complex. In each of twelve different concentrations of each substrate the DRT activity of V2 was measured with the thiobarbituric acid method and the substrate saturation curves plotted. 

In the case of the oligomeric enzymes the kinetic cooperativity is more complex. The interactions between the subunits can influence the rate (speed) of the transition or even alter the three-dimensional structure of the subunits themselves. Weakly coupled subunits generate no sigmoidal substrate saturation curve and in this instance the kinetic cooperativity can be greater or smaller than the corresponding substrate binding cooperativity. This is the case for V2, as it can be seen from the values of h exf(13)-... 

In other words, a sigmoidal substrate saturation curve that fits the velocity equation of the former model can be shown to fit the velocity equation of the latter model as weU. 

Consequently, substrate saturation curves are sigmoid instead of hyperbolic. CTP exerts its inhibitory effect by increasing the interaction between the four catalytic binding sites, which decreases the affinity of the enzyme for the substrate. 

The rate of PP-ribose P synthesis in the dialyzed supernatant increases almost linearly with ribose-5-P concentration in the absence of ATP and of an ATP regenerating system (Fig. lA). There is nearly no PP-ribose-P synthesized from ribose-l-P under these conditions (Fig. IB). In the presence of phosphoenolpyruvate and pyruvate kinase, but without any addition of ATP, both substrate saturation curves are hyperbolic. The apparent Km of PP-ribose-P synthetase for ribose-5-P is 224 57 yM, and the apparent V ax is 48.7 5.6 nmoles per h per mg protein. Similar values are measured in the presence of ribose-l-P. However, it is not a substrate for the PP-ribose-P S3mthetase purified from rat liver (1). Ribose-l-P is probably converted first to ribose-5-P by a phosphoribomutase which is present in rat liver (4). 

The mutant and the normal enzyme were found to have similar phosphate activation curves (Pig. 2) and similar substrate saturation curves for R-5-P and for ATP. 

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