Biphasic saturation kinetics

Iron(III)-pyrophosphate looks promising as an alternative to iron(III)-carbohydrate preparations for parenteral administration for treatment of anemia.Kinetics of removal of iron from transferrin (tf) by pyrophosphate (pp) were found to be biphasic under certain conditions, with the rapid first phase attributed to the formation of a pp—Fe—tf—CO intermediate.A later study of the kinetics of removal of iron from transferrin employed pyrophosphate and tripodal phosphonates such as nitrilotris(methylenephosphonic acid), N(CH2P03H2)3. For the tripodal ligands there are parallel first-order and saturation pathways, with the latter dominant (contrast the exclusively first-order reaction of ferritin with nitrilotriacetate) for pyrophosphate the paths are roughly equal in importance. The saturation kinetics suggest that tfiFe-phosphonate intermediates play an important role in the kinetics. 

Kmi would be the standard Michaelis constant for the binding of the first substrate, if [ESS] = 0. Km2 would be the standard Michaelis constant for the binding of the second substrate, if [E] = 0 (i.e., the first binding site is saturated). In the complete equation, these constants are not true Km values, but their form (i.e., Km] = (k2 + k25)/k 2) and significance are analogous. Likewise, k25 and k35 are Vmi/Et and Vm2/Et terms when the enzyme is saturated with one and two substrate molecules, respectively. Equation (10) describes several non-Michaelis-Menten kinetic profiles. Autoactivation (sigmoidal saturation curve) occurs when k35 > k24 or Km2 < Km 1, substrate inhibition occurs when k24 > 35, and a biphasic saturation... 

A second type of nonhyperbolic saturation kinetics became apparent during studies on the metabolism of naproxen to desmethylnaproxen (32). Studies with human liver microsomes showed that naproxen metabolism has biphasic kinetics and is activated by dapsone (T. Tracy, unpublished results). The unactivated data shows what appears to be a typical concentration profile for metabolism by at least two different enzymes. However, a similar biphasic profile was obtained with expressed enzyme (25). This biphasic kinetic profile is observed with the two-substrate model when V/rn2 > Eml and Kml Km2. The appropriate equation for the two-site model when [S] < Kml is... 

Biphasic Kinetics (Nonasymptotic) For the purposes of this discussion, a biphasic kinetic profile is defined as one in which the kinetic profile does not follow saturation kinetics and has two distinct phases (Fig. 4.6). Note that sigmoidal kinetics may also be biphasic but exhibits saturation. 

The biphasic kinetic pattern described for the removal of iron from transferrin by pyrophosphate can be ascribed to the two different iron-containing sites in transferrin/ Various anions and acids can assist such removal/ Details of iron removal have been probed by studying the kinetics of metal removal from transferrin derivatives containing iron and cobalt variously distributed between the two inequivalent binding sites, and from transferrins containing iron in only one of the two sites. The kinetics of iron removal from the Fee sites show a first-order dependence on pyrophosphate concentration, from the FeN sites show saturation kinetics. The current situation with respect to mechanisms of iron removal from, and incorporation into, transferrin have been reviewed. ... 

Even cursory inspection of typical (v,[) data shows tliat tire evolution does not follow tire single exponential approach to saturation implied by, for example, (equation C2.14.22) witli initial concentrations Xq Such data are sometimes described as biphasic , and one encounters attempts to fit and inteiyDret tliem witli two exponentials, even tliough tliere does not seem to be any tlieoretical justification for doing so. The basic kinetics of adsorjDtion are described by ... 

In the meantime temperature-dependent stopped-flow measurements were conducted on the latter complex in order to determine the activation parameters of the N-N cleavage reaction (24). Plots of the absorption intensity at 418 nm vs. time at T — —35 to +15°C indicate biphasic kinetics with two rate constants 0bs(p and obs(2)> in analogy to our measurements of the tungsten complex. This time, however, both rates depended upon the acid concentration. Interestingly much smaller rate constants 0bs(i) and 0bs(2)> were found for all acid concentrations than given by Henderson et al. for his (single) rate constant kobs (up to 1 order of magnitude). Furthermore plots of 0bs(i) and kohs(2) vs. the acid concentration showed no saturation behavior but linear dependencies with slopes k and k and intercepts k und k, respectively (s — acid dependent and i — acid independent), Eq. (2) ... 

Mean clearance (CL) values for cetuximab are displayed as a function of dose in Fig. 14.3. Mean CL values decreased from 0.079 to 0.018 L/h/m2 after single cetuximab doses of 20 to 500 mg/m2, respectively. In the dose range 20 to 200 mg/m2, CL values decreased with dose. At doses of 200 mg/m2 and greater, CL values leveled off at a value of approximately 0.02 L/h/m2. This biphasic behavior suggests the existence of two elimination pathways. The elimination of cetuximab apparently involves a specific, capacity-limited elimination process that is saturable at therapeutic concentrations, in parallel with a nonspecific first-order elimination process that is non-saturable at therapeutic concentrations. Increasing doses of cetuximab will therefore ultimately lead to the saturation of the elimination process that is capacity-limited and that follows Michaelis-Menten kinetics, whereas the first-order process will become the dominant mechanism of elimination beyond a particular dose range. 

Paclitaxel has non-linear kinetics peak plasma concentrations and drug exposure increase disproportionately with increasing doses and the pharmacokinetics depend on the schedule of administration. Saturation is reached with high-dose short infusions (4). Paclitaxel has been reported to follow both biphasic (5) and triphasic models (6). The half-life has been estimated at 6-13 hours after intravenous administration (7). 

FIGURE 4.6 Representative plot depicting biphasic kinetics (two phases with one not achieving saturation). 

FIGURE 4.9 Eadie-Hofstee plots useful to diagnose the type of kinetics occurring in a reaction for (a) hyperbolic (Michaelis-Menen) kinetics, (b) Sigmoidal kinetics, (c) Biphasic kinetics with no saturation of second phase, and (d) Substrate inhibition kinetics. 

The sorption of mono-CB on marine sediment required 3 hours to reach a sorp-tion/desorption equiUbration (Zhao et al, 2001). Compared with sediment organic carbon content, surface and microporosity of the sediment might have a more important effect on the sorption of mono-CB (Zhao et al, 2001). Temperature does not have great influence on the adsorption behavior of mono-CB in seawater, but the saturate sorption capacity decreases as the temperature increases. The Tenax-mediated desorption of HCB from four freshly spiked, artificial sediments exhibited biphasic kinetics the first-order rate constants of the fast desorbing phase and slow desorbing phase were 9.6 x 10 and 7.2 x lO h respectively (Chai et al, 2007). The fast desorption fractions for the four sediments varied from 41.2 to 68.8% (Chai et al, 2007). 

An analysis of the kinetics and product distribution of ketonization in D2O saturated nitrobenzene gave interesting insight into the mechanism of inner phase ketonization (Figure 9.33). Ketonization showed biphasic kinetics and is initiated by deprotonation of 105 (I II). Ion pair II either collapses back to 105 (I) or forward to acetophenone (III). 

The isolated PSn rc preparation shows a spin polarised triplet EPR spectrum with polarisation AEEAAE as expected (Fig.2). The signal intensity was optimal only after addition of dithionite, presumably ensuring that P680 was not partially oxidised. The intensity had a microwave power optimum at 100 jiW at 5 K, although at this power level the kinetics were biphasic. The EPR signal intensity did not show light-saturation even at 300 mW excitation intensity, but at >100 mW there is a distortion of the x peak kinetics(Fig.3A), so that low light conditions are preferable. 

Purified cottonseed NAPE synthase enzyme exhibited non-Michaelis-Menten biphasic kinetics with respect to the free fatty acid substrates, palmitic and linoleic acids. Kinetic parameters for the two saturable sites were calculated from various transformations e.g., double-reciprocal and Hill plots Cornish-Bowden, 1995) of initial velocity/ substrate concentration data and are summarized in TABLE 1. Preliminary experiments with several group-specific modifiers indicated that NAPE synthase was progressively inactivated by increasing concentrations of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), diisopropyl fluorophosphate (DFP), phenylmethylsulfonylfluoride (PMSF), diethylpyrocarbonate (DEPC) (TABLE 2). These results suggest that NAPE synthase may form a thioester- or ester-intermediate through a cysteine or serine residue, respectively, and a histidine residue may participate in catalysis as well. 

A series of ynenol lactones (stmcture 2) were studied as inhibitors of human leukocyte elastase (Tam et al., 1984 Spencer et al., 1986 Copp et al., 1987). Some of the compounds were alternate substrate inhibitors, being hydrolyzed by the enzyme to the reactive I but then deacylat-ing without an inactivation step. However, with the compound 3-benzyl ynenol butyrolactone (stmcture 2, where R = benzyl, R = H), the acyl-enzyme (E-I ) was stable enough to allow the second alkylation step, resulting in inactivated enzyme. All kinetic constants were determined. Continuous assays gave biphasic kinetics, the second minor phase possibly due to the presence of isozymes or enantiomers of the inhibitor. Immediate diffusion-limited inhibition was observed and gave a competitive Ki value of 4.3 0.7 xM. The first phase of inhibition was saturable, and analysis of the rates gave = 0.090 0.007 s , and... 

The latter observation corresponds well with the finding, that under comparable conditions pyruvate uptake by pea chloroplasts showed biphasic kinetics following a saturation at lower (<1 mM) but a diffusion mechanism at higher (>1 mM) concentrations (Proudlove and Thurman, 1981). The portion of label in coenzyme A-bound fatty acids made up about 16% after incubation with 1- C-acetate and about 30% after 2- C-pyruvate incorporation. 

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