Enzymes saturation

Reaction Potentials. The reaction potentials Vm. Vq and Vr are the rates at which methanogenesis, CH4 oxidation and oxic respiration would proceed in situ were all enzymes saturated with the necessary substrates. They depend on in situ enzyme concentrations and hence on in situ microbial populations. They change over time. 

In the design of coatings, hydrogels and matrices based on azo polymers, the number of azo bonds in the polymers should not be too high, as this could lead to enzyme-saturated conditions slowing down the degradation process and thus drug release [72]. 

For the ATPase activity of the enzyme, Mg2+ is required. Kinetic studies confirm that MgATP is the substrate and that while free ATP binds to the enzyme it inhibits activity. It appears that MgATP binds at high and low affinity sites, with Km values about 3 p,mol dm-3 and 0.2 mmol dm-3 respectively. For phosphorylation of the enzyme, saturation of the high affinity site is sufficient for maximum activity. Binding at the low affinity sites inhibits ADP/ATP exchange and may represent a modulator role for these sites. This low affinity site may be the site for the substrate in the K+-phosphatase reaction. 

The amount (k.E) is the maximum reaction rate (n. jmax), that always occurs at conditions where limiting substrate concentrations are much higher than the constant km (S > > km), allowing enzyme saturation by substrate. 

Figure 5.3 Phosphorylation-dephosphorylation cycle activation as a function of the activating signal 6 and available free energy y. The solid lines and dashed lines are without and with enzyme saturation, i.e., Equations (5.6) and (5.18), respectively. In both cases, from top to bottom y = 1010,104, and 103. All computations are done with /x = 0.001, and for the dashed lines = - = 0.01. If y = 1, then both the solid and dashed lines will be strictly horizontal.

For example, for y1 = = 0.01, n = 51, indicating an ultrasensitive swi tch The coefficient is large if K and K <enzyme reactions are highly saturated. This means the rates for the phosphorylation and dephosphorylation reactions S S are independent of the respective substrate concentrations [S] and [S ]. Hence, both reactions are effectively zeroth order. Ultrasensitivity arises from this zeroth-order behavior. In comparison with the case where there is no enzyme saturation, the dashed lines in Figure 5.3 show the sharp transitions. Figure 5.3 also shows that the ultrasensitivity does not change the amplitude of the switch. 

Metabolic loading tests and the determination of enzyme saturation with cofactor measure the ability of an individual to meet his or her idiosyncratic requirements from a given intake, and, therefore, give a nearly absolute indication of nutritional status, without the need to refer to population reference ranges. A number of factors other than vitamin intake or adequacy can affect responses to metabolic loading tests. This is a particular problem with the tryptophan load test for vitamin Be nutritional status (Section 9.5.4) a number of drugs can have metabolic effects that resemble those seen in vitamin deficiency or depletion, whether or not they cause functional deficiency. 

To determine the Ki and kmact values, first a plot of the log of the enzyme activity versus time is constructed (Fig. 16a). The rate of inactivation is proportional to low concentrations of the inactivator, but becomes independent at high concentrations. In these cases, the inactivator reaches enzyme saturation (just as substrate saturation occurs during catalytic turnover). Once all of the enzyme molecules are in the E-I complex, the addition of more inactivator does not affect the rate of the inactivation reaction. The half-lives for inactivation (fi/2) at each inactivator concentration (lines a-e in Fig. 16a) are determined. The fi/2 at any inactivator concentration equals log 2/kina( t,appf in the limiting case of infinite inactivator concentration, fi/2 = 0.693/kiiiact (log 2 = 0.693). A replot of these half-lives versus the inverse of the inactivator concentration, referred to as a Kitz and Wilson replot, is constructed to obtain the K and kjuact values (Fig. 16b). 

The effects of this concentration gradient are most significant at low bulk concentrations of the substrate, since substrate is converted to product as soon as it reaches the surface of the particle, so that the surface concentration of substrate is zero. At very high bulk substrate concentrations, the enzymatic reaction rate is limited by enzyme kinetics rather than mass transport, so that surface concentrations do not differ significantly from those in the bulk. Because of the concentration gradient, however, enzyme saturation with substrate occurs at much higher bulk substrate concentrations than required to saturate the soluble enzyme. Apparent Km values (K m) for immobilized enzymes are larger than Km values obtained for the native soluble enzymes. 

Requirement of K+ by fructokinase has been demonstrated.312,319 The K+ ion acts as an allosteric activator, and it cooperatively increases the reaction velocity.320 The Mg2+ ion is also important, as the activity of fructokinase depends on the ratios of ATP4- MgATP2- and of KATP3- MgATP2-. The optimal ratios vary, depending upon the enzyme saturation and the K+ concentration.320... 

At high substrate concentrations, when S Ks, the kinetics reach the enzyme saturated rate and become zero-order ... 

The rate of a-chymotrypsin-catalyzed hydrolysis as a function of overall GPANA concentration in CTAB reversed micelles and in aqueous solution are shown in Figure 5. It is apparent that the reaction rate in the reversed micellar solution is on the order of 50 times more rapid than in the aqueous system. Furthermore, in the reversed micellar system there is no indication of enzyme saturation as the reaction is first order in substrate concentration. As enzyme saturation kinetics are not observed, it is impossible to differentiate between the parameters kcat and Kg. Instead a second order bimolecular rate constant for both the micelle interior ( micelle) and for what is experimentally observed ( observed) is defined. 

At concentration values above Ks it is not enzyme saturation but the catalytic turnover of the enzyme that becomes more and more limiting. Being proportional to the concentration of the ES complex, the reaction rate cannot rise above the vmax value when all enzyme molecules are in the ES form. The enzyme kinetics turn to zero order. 

As discussed above, the most common indicator of enzyme activity is KM, which is determined by measuring Vmas at enzyme saturation conditions and Ve at a concentration of S, [S], near to the saturation concentration of S for a given concentration of E, [E]. V0 is taken as the initial slope of the plot of reaction conversion vs time for that value of [S], and a series of such plots and V values are obtained at different values of [E], The slopes of a plot of Ve vs [E] then gives VC/[E], which can be used to calculate KM from the Michaelis-Menten equation. 

The reaction is reversible and can be employed in either direction. In the forward reaction, serum containing an unknown amount of LDH would be added to a solution containing enzyme saturating concentrations of lactic acid and NAD, and the increase in absorbance at 340 nm would be measured as a function of time. 

FIGURE 1.7 Graphical representation of three concentrations of drug over time for which drug levels above the dotted line show enzyme saturation and hence nonlinearity. 

The dimensionless saturation factor represents a measure of enzyme saturation and allows for the approximation of the Michaelis-Menten rate, either to a first order (0 1) or to zero order (0 1). 

---