Substrate affinity constants

At substrate concentrations lower than the Michaelis-Menten constant, also known as the substrate affinity constant, less than half of the enzyme population would be expected to have substrate associated with it (Figure 1). 

Fig. 3. Double reciprocal plots of a) competitive inhibition, where introduction of the inhibitor produces changes exclusively in the substrate affinity constant (Km) and b) noncompetitive inhibition, where inhibition is observed as a decrease in the maximum velocity of the enzyme catalyzed (Vm x) reaction...

Estimations of enzyme kinetic constants have been used previously to explain the substrate selectivity exhibited by pea and spinach stromal ATI (Frentzen, eta/., 1983). We have determined the acyl substrate affinity constants and maximal velocities for the enzymes isolated from chloroplasts of three chilling sensitive species and will rely upon previously published values for spinach and pea for our discussion. We have not determined Km values for G3P in tne presence of acyl-ACPs for any of these enzymes. 

In non-competitive inhibition, the substrate (S) and inhibitor (I) have equal potential to bind to the free enzyme (E). The inhibitor forms a ternary complex with enzyme-substrate (ES) whereas the substrate will form another ternary complex with enzyme-inhibitor (El). Since the non-competitive inhibitor had no effect on the binding of substrate to the enzyme, the Km value remained consistent (or unchanged). There are two different ways for the formation of ESI ternary complex this complex would not form the product and therefore was decreased. Non-competitive inhibitor had no effect on substrate binding or the enzyme-substrate affinity, therefore the apparent rate constant (K ) was unchanged.5 A possible reason for product inhibition was because of the nature of 2-ethoxyethanol,... 

The two substrate kinetics of the overall reaction catalyzed by the complex in permeabilized membranes showed classical ping-pong kinetics in accordance with a phosphorylated enzyme intermediate. The affinity constants for fructose and P-enolpyruvate were 8 and 25 /iM, respectively. 

Important inherent characteristics of an enzyme that should be considered are the substrate affinity, characterized by the Michaelis constant the rate of turnover fecat> providing the catalytic efficiency fecat/ M. and the catalytic potential. Several attempts to compare enzyme catalysis with that of platinum have been published. Direct comparisons are difficult, because enzyme electrodes must be operated in aqueous electrolyte containing dissolved substrate, whereas precious metal electrodes aie often supplied with a humidified gaseous stream of fuel or oxidant, and produce water as steam. It is not straightforward to compare tme optimal turnover rates per active site, as it is often unclear how many active sites are being engaged in a film of enzyme on an electrode. 

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

It is perfectly possible for some substrate-modulator combinations to result in an increase in substrate affinity, an increase in the rate of product formation, or both. The same analytical approaches may be used to study such compounds as have been described earlier to assess inhibitory mechanisms and potencies. However, with an allosteric activator, the dissociation constant might better be termed and values for a and p are more likely to be less than one, and greater than one, respectively. As is the case for inhibition, allosteric enzyme activation would be expected to exhibit substrate dependence (Holt et al., 2004). 

Although it is not known whether the base is an essential feature of a substrate, the nucleoside moiety is necessary since bis- and tris-p-nitrophenyl phosphate esters are not hydrolyzed (61). The nature of the R substitution on the 3 -OH clearly affects the affinity of the substrate or inhibitor for the enzyme, even though it does not affect the maximal catalytic rate constant (Table I). The importance of the 5 - and 3 -phosphate groups in determining the affinity of inhibitors (3, 66) is consistent with the contribution of these groups to substrate affinity (61). These effects result from the phosphoryl groups themselves rather than... 

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