INDEX substrate binding

The catalytic efficiency of an enzyme is indicated by its kcatIKM value, the value combining the effectiveness of both the productive substrate binding and the subsequent conversion of substrate molecules into product (Copeland, 2000). This value is the apparent second-order rate constant for enzyme action under conditions in which the binding site of the enzyme is largely unoccupied by substrate. The kcatIKM value is the index for comparing the relative rates of cleavage of alternative, competing substrates. The KM is the Michaelis constant, an apparent dissociation constant and hence a measure of substrate affinity. This value equals the concentration of substrate needed to reach half maximum velocity of the enzyme reaction. 

The Hill coefficient is an index of the cooperativity in the substrate binding process—the greater the value of n, the higher the cooperativity. For the case where n = 1 (no cooperativity), the Hill equation reduces to the Michaehs-Menten model. If the cooperativity of the sites is low, n will not correspond to the number of substrate-binding sites, but the minimum number of effective substrate-binding sites. Regardless of this limitation, the Hill equation can still be used to characterize the kinetic behavior of a cooperative enzyme. In this case, n becomes merely an index of cooperativity, which can have noninteger values. 

The advantages of SPR experiments are that only small amounts of sample are required,72 often hundreds of microliters of solutions with nanomolar to micromolar concentration of reactants and the substrate attached to the surface can oftentimes be reused after washing in buffer. The fact that changes in the refractive index values are measured avoids the need to use absorption or fluorescence markers to follow the binding kinetics. 

The RUI method is a modification of the brain uptake index method [29] which was first described by Aim and Tornquist [4], The RUI approach consists of a single-arterial (e.g., intracarotid) injection technique, where the primary objective is to analyze the influx of the test substrate from the circulating blood to the retina through the BRB (i.e., blood-to-retina direction). This approach, for example, has been used to determine the retinal uptake of the test substrate which has a relatively high permeability across the BRB. The advantage of this approach is that it avoids the effect of plasma-protein binding of the test substrate and allows the retinal uptake of the test substrate... 

Inhibition of the initial step of a biosynthetic pathway by an end product of the pathway is a recurrent theme in metabolic regulation. In addition, many key enzymes are regulated by ATP, adenosine diphosphate (ADP), AMP, or inorganic phosphate ion (Pi). The concentrations of these materials provide a cell with an index of whether energy is abundant or in short supply. Because ATP, ADP, AMP, or P often are chemically unrelated to the substrate of the enzyme that must be regulated, they usually bind to an allosteric site rather than to the active site. 

Fig. 6 Resonance angles on an SFIO substrate with a refractive index of (a) a bulk environment n nv ( conventional SPR, LSPR with /I = 50 nm, and A LSPR with A = 100 nm) and (b) a target binding layer nsAM- (filled dsAM = 1 nm and empty dsAM = 3 nm) Reprinted from [24], Copyright (2008), with permission from the Optical Society of America...

Figure 3. Reaction scheme of complementary replication of single-stranded RNA. Reaction consists of four phases initiation, elongation, product release, and template reactivation. Reaction product (replica) is complementary to template. Substrates are four nucleoside triphosphates ATP, GTP, UTP, and CTP. Pyrophosphate (pp) is waste product at each step of incorporation. Symbols /, RNA template chain E, enzyme (replicase) P, growing RNA replica chain. Indexes A, association D, dissociaton S, substrate F, phosphate diester bond formation PR, product release the numbers 3, or 5, refer to end of the RNA chain to which the enzyme binds or from which it dissociates (cf. ref. 10).

The Michaehs constant is a fundamental characteristic of an enzyme that incorporates important information about the enzyme. First, it gives the concentration of substrate necessary to bind to half of the available sites on the enzyme. Second, it gives an index of the relative binding affinity of the substrate to the active sites on the enzyme. Michaehs constants have been determined and tabulated for a large number of enzymes. Since is actually a concentration, the values are usually expressed in mM units. 

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