Specificity competing substrates

The quantity kcat/Km is a rate constant that refers to the overall conversion of substrate into product. The ultimate limit to the value of k at/Km is therefore set by the rate constant for the initial formation of the ES complex. This rate cannot be faster than the diffusion-controlled encounter of an enzyme and its substrate, which is between 10 to 10 per mole per second. The quantity kcat/Km is sometimes called the specificity constant because it describes the specificity of an enzyme for competing substrates. As we shall see, it is a useful quantity for kinetic comparison of mutant proteins. 

Specificity, in the sense of discrimination between competing substrates, is independent of the above three effects. The reasons are discussed in detail in Chapter 13. The basic reason is that specificity depends on kcal/KM, and strain and nonproductive binding do not affect the value of kcJKM because it is independent of interactions in the ES complex (equations 12.10 and 3.36). Equation 12.16 shows that induced fit does alter kcal/KM for the active conformation, but equally for all substrates (i.e., by a factor of K). 

It was shown in Chapter 3, section G2, that specificity for competing substrates is controlled by kcatIKM. If the rate of reaction of the specific substrate A is vA, and that of the competitor B is vB, then... 

It will be shown later (equation 3.41) that this result holds at any substrate concentration. It will also be shown later (equation 3.44) that kcJKM determines the specificity for competing substrates. For this reason, kc.JKM is sometimes referred to as the specificity constant. ... 

The important conclusion is that specificity, in the sense of discrimination between two competing substrates, is determined by the ratios of kcJKM and not by Ku alone. Since km/KM is unaffected by nonproductive binding (section E) and by the accumulation of intermediates (section F), these phenomena do not affect specificity (see Chapter 13). Note that equation 3.44 holds at all concentrations of substrates. 

Specificity between competing substrates depends on the relative binding of their transition states to the enzyme. Enzyme-transition state complementarity maximizes specificity because it ensures the optimal binding of the desired transition state. This is also the criterion for the optimal value of kcatIKM, which is not surprising, since specificity is determined by kcatIKM. Maximization of rate... 

We now introduce a new specificity term called the selectivity, S, which describes the overall specificity for competing substrates when editing is talking place. S is defined by... 

Exposure to two or more chemical agents can result in altered expression of hepatotoxicity. However, qualitatively different interactions may be achieved, depending upon the relative timing of exposures. Simultaneous exposure to competing substrates of a specific CYP isozyme will often slow metabolism and can be protective against reactive metabolite-mediated hepatotoxicity. Alternately, pretreatment with one agent may induce metabolic enzymes that either protect against or potentiate... 

Another parameter often referred to when discussing Michaelis-Menten kinetics is kcaJ Ky. This is an apparent second-order rate constant that relates the reaction rate to the free (not total) enzyme concentration. As described above, at very low substrate concentrations when the enzyme is predominantly unbound, the velocity (f) is equal to [El Ky. The value of Is JKy sets a lower limit on the rate constant for the association of enzyme and substrate. It is sometimes referred to as the specificity constant because it determines the specificity of the enzyme for competing substrates. 

Competing substrates. Suppose that two substrates, A and B, compete for an enzyme. Derive an expression relating the ratio of the rates of utilization of A and B, VpJVg, to the concentrations of these substrates and their values of k 2 and K iy[. (Hint Express as a function of k fK for substrate A, and do the same for Eg.) Is specificity determined by if alone ... 

Because adsorption equilibria can produce a substantially different population at the semiconductor-liquid interface than is present in solution, interfacial charge trapping can produce specific activation of the better adsorbate from a mixture. Thus adsorption pre-equilibria are likely to be important in controlling the relative rates of photooxidation of competing substrates on semiconductor surfaces. Because adsorption equilibria can be influenced by the addition of very small amounts of cosolvent additives, higher reactivity [36] and higher selectivity can often be simulta-... 

Prior to MS-based substrate specificity assays, certain NRPS substrate specificities can be predicted by bioinformatics. Adenylation domain substrates can be predicted based on their 10 letter code 99,100 by substrate prediction tools such as the NRPS predictor.101 Methyltransferases can be predicted in their substrates and methylation sites by bioinformatic analysis too.102 In addition, substrates of catalytic NRPS domains and tailoring enzymes can be predicted by the structure of the known NRP natural product. Either way, predicted substrates of NRPS domains need to be experimentally verified. A traditional technique to determine substrate specificity of an A domain is the adeonsine triphosphate-pyrophosphate (ATP-PP ) exchange assay. The ATP-PP exchange assay characterizes substrates indirectly by observing the radioactive pyrophosphate incorporation into ATP from a reverse reaction with pyrophosphate and the acyl-adenylate of the substrate.103 Because the PP exchange measures the back exchange of pyrophosphate into ATP, the determined substrate can deviate from the true substrate as it may be only the kinetically most competent substrate of the reverse adenylation reaction. In contrast to this assay, MS has become a more reliable tool to identify NRPS substrates because it determines the true substrate specificity by detection of the complete adenylation reaction product, that is, the substrate tethered on a T domain. 

Specificity between competing substrates is therefore given simply by relative values... 

Provided that the substrates being compared have sufficient binding energy to compensate for the unfavourable conformational change of the enzyme, then induced fit cannot explain specificity between competing substrates. Compared with the situation where the enzyme is initially in the active conformation, the induced fit mechanism reduces the value of for all substrates by the same fraction and... 

Specificity is a fundamental property of eirzymes, but it is often assessed in an unsatisfactory way, by comparing the kinetic parameters for different reactions measured in isolation from one another. In the cell, specificity must clearly refer to the capacity of an enzyme to react selectively with one substrate when others are present simultaneously, and any satisfactory measure of specificity should take account of this (8). The equation for reaction of one substrate A in the presence of a competing substrate A follows a form similar to that for competitive inhibition, equation 15 ... 

Furthermore, in vivo specificity results from a competition between sub-strates/inhibitors for the active site of the enzyme. The important parameter then becomes the free energy of activation as measured by 2 - Specificity between competing substrates is given by the relative values of k- K and not by the individual values of 2 or K. In fact k2K varies by up to 10 over a range of P-lactams with the R39 and R61 transpeptidase enzymes (Frere and Joris, 1985). This variation emphasises one of the difficulties of finding relationships between structure, chemical reactivity and activity towards enzymes. 

Obtaining accurate measurements of Km is important because Km provides a quantitative measure of enzyme-substrate complementarity in binding (when Km Ks), and such values can be used to compare relative affinities of competing substrates. Second, the combined determination of Vmax (oc cat) and Km for competing substrates provides for a quantitative comparison of specificity (selectivity) of the enzyme among substrates through the use of the specificity constant, or VmaxZ-K m [Eq. (14.4)] (Fersht, 1985). 

The kJK is the kinetic parameter (an apparent second-order rate constant) that is most appropriate for distinguishing substrate specificity. Kinetically, specificity means the capability of an enzyme to discriminate between competing substrates and is a function of both and... 

EH-catalyzed hydrolysis of a racemic epoxide, resulting in the formation of a vicinal diol with an enantiomeric excess of 100%>eep>0% at complete conversion (c=100%) of the substrate. Depending on the EH-substrate interactions and consequently its substrate-related enantiomeric ratio or f-value (which characterizes the ability of the enzyme to discriminate between the two competing substrate enantiomers [1]), enantiopure epoxide are obtained at a specific degree of conversion within the range of 50%rapid hydrolysis of the preferred epoxide enantiomer, followed by a much slower hydrolysis of the remaining epoxide. 

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