Heart kinetic constants

A careful kinetic study has shown that, although all catalyse the same reaction, both the kinetic constants Km and Km, differ. The kinetic characteristics match the requirements of the tissues, e.g. Fmax is high in skeletal muscle but low in heart muscle. 

Negative cooperativity of nucleotide binding (K ) and positive cooperativity of catalysis (E , ) between catalytic sites of Fj (kinetic constants for ATP hydrolysis at multiple catalytic sites of beef heart MFj)... 

For a proper understanding of the role of the translocators in the regulation of metabolism, knowledge of their kinetic constants is indispensable. Table 1 summarises these parameters. Because of technical difficulties, in most cases it has not been possible to determine these parameters at 37°C. It is also important to stress that the kinetic constants have been determined in isolated mitochondria. It is likely that the kinetic constants in the intact cell are different, one reason being that there is an inhibitory interaction of cytosolic anions with the various translocators. Some of these effects are given in Table 2. With the exception of a few cases (the a-oxoglutarate translocator in heart [18] and the carnitine and aspartate translocators see Sections If, iii and iv), little is known about the values of the metabolites to be transported from the matrix side of the mitochondrial membrane. In the case of citrate and ATP transport such information is difficult to obtain because most of the intramitochondrial citrate and ATP is chelated with Mg " and only the free anions are transported. Likewise, little is known about possible competition between metabolites present in the matrix for export out of the mitochondria. The complexity of the complete kinetic analysis of a translocator, in which both the external and internal concentrations have been taken into account, is illustrated by the studies of Sluse et al. [18] on a-oxoglutarate transport in heart mitochondria. 

Table 16. Kinetic Constants for UftakEj and UptakEj in the Rat Heart...

The kinetics of intramolecular electron transfer from Ru(II) to Fe(III) in ruthenium-modified cytochrome c has been studied [77-80]. In these studies electron transfer from electron-excited Ru(II) (bpy)3, which was added to the protein solution, to ruthenium-modified horse heart cytochrome c, (NH3)5Ru(III) (His-33)cyt(Fe(III)), was found to produce (NH3)5Ru(II) (His-33)cyt (Fe(III)) in fivefold excess to (NH3)5Ru(III) (His-33)cyt(Fe(II)). As in refs. 72 and 73, in the presence of EDTA the (NH3)5Ru(II)(His-33)cyt(Fe(III)) decays mainly by intramolecular electron transfer to (NH3)5Ru(III)(His-33)cyt(Fe(II)). The rate constant k — 30 3s 1 at 296 K and does not vary substantially over the temperature range 273-353 K. Above 353 K intramolecular Ru(II) - Fe(III) electron transfer was not observed owing to the displacement of methionine-80 from the iron coordination sphere. The distance of intramolecular electron transfer in this case is also equal to 11.8 A (see Fig. 19). 

Detailed kinetics of ATP hydrolysis in single-site (uni-site) and steady-state (multi-site) conditions by beef heart Fi were studied extensively (Fig. 11.5).45 46) At an ATP/ Fi ratio of less than 1, ATP binds to a catalytic site and is hydrolyzed slowly (uni-site catalysis). The equilibrium constant between bound substrate (ATP) and products (ADP and Pi) bound at the catalytic site of Fi was close to 1, indicating that the equilibrium can occur without change in free energy. In the presence of excess ATP (multi-site catalysis), ATP binds to all three catalytic sites, and the ATP at the first site is hydrolyzed at a rate that is at least 106 times higher than is the case in uni-site catalysis. 

The initial rate equation is again of the form of Eq. (1) with the kinetic coefficients as in Table I, which shows that the mechanism differs from the simple ordered mechanism in three important respects. First, the isomerization steps are potentially rate-limiting evidence for such a rate-limiting step not attributable to product dissociation or the hydride-transfer step (fc) has been put forward for pig heart lactate dehydrogenase 25). Second, Eqs. (5) and (6) no longer apply in each case the function of kinetic coefficients will be smaller than the individual velocity constant (Table I). Third, because < ab/ a< b is smaller than it may also be smaller than the maximum specific rate of the reverse reaction that is, one of the maximum rate relations in Eq. (7) need not hold 26). This mechanism was in fact first suggested to account for anomalous maximum rate relations obtained with dehydrogenases for which there was other evidence for an ordered mechanism 27-29). 

Calculation of model output. At the heart of a model simulation is the calculation itself. Usually this code is in a loop over time or distance. Considerable attention must be paid to the fitness of the model, both in terms of its underlying assumptions and its coding as a computer program. It must be understood that a model is not reality. Where the model deviates from reality must be known, and the computer realization of the model must not allow nonphysical behavior. For example, in fitting kinetic data rate constants cannot... 

In an experiment with baby rabbit heart muscle tissue [6], the following kinetic results were obtained (a) for creatine phosphate, CrP, of concentration 1.8 mM the apparent reaction rate was 1.75 xM min and (b) for CrP of concentration 0.35 mM the apparent rate was 0.80 xM min . Calculate the Michaelis constant, /Cm, and the maximum reaction rate, l/max, for this reaction. 

Sutin s kinetic studies on the oxidation of horse-heart ferrocytochrome c by tris-(phen)cobalt(iii) have recently been extended to acid pH. The reaction is first-order with respect to each reactant but the dependence of the rate on [H+] is not simple. Measurements in chloride medium (7=0.13 mol 1 ) over the pH range 1—7 revealed a rate maximum at pH 2.9 (A =6.7x 10 1 mol" s at 25 °C). By contrast, the rate constants at pH 1.0 and 5.8 are 3.2 x 10 and 2.1 x 10 1 mol" S", respectively. Below pH 1.7, biphasic kinetics are observed, the slower reaction having a rate constant of ca. 2 s" (independent of oxidant concentration). The slow process is ascribed to a conformational change in the ferricytochrome c which is produced in... 

The kinetics of oxidation of a trifluoroacetylated derivative of horse-heart ferro-cytochrome c by hexacyanoferrate(iii) have been measured as a function of temperature a rate constant (25 °C and 7=0.1 mol 1 ) of 3.0 x 10 1 mol" s was obtained, with H =0.4 kcal mol and = — 33 cal mol . The low value of A77 with the modified cytochrome (which does not bind the oxidizing agent) is similar to that found with the native protein (which does) and it is suggested that in the former case the mechanism involves a pre-equilibrium between two forms of the cytochrome, one of which is more easily oxidized. The reaction of [nitrotyrosyl] cytochrome c with imidazole is biphasic and the kinetics are consistent with the existence of a similar equilibrium involving two forms of the cytochrome. 

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