Enzyme specificity constant

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. 

The enantioselectivity of biocatalytic reactions is normally expressed as the enantiomeric ratio or the E value [la], a biochemical constant intrinsic to each enzyme that, contrary to enantiomeric excess, is independent of the extent of conversion. In an enzymatic resolution of a racemic substrate, the E value can be considered equal to the ratio of the rates of reaction for the two enantiomers, when the conversion is close to zero. More precisely, the value is defined as the ratio between the specificity constants (k st/Ku) for tho two enantiomers and can be obtained by determination of the k<-at and Km of a given enzyme for the two individual enantiomers. 

As an example, we mention the enzyme catalase, which catalyzes the decomposition of H2O2 to H2O and O2 at a turnover number of kcat = 10 and a high specificity constant of kcat/f M = 4 x 10 mol s . Such activities are orders of magnitude higher than those of heterogeneous catalysts. 

Specificity constant Defined as kcJKm. It is a pseudo-second-order rate constant which, in theory, would be the actual rate constant if formation of the enzyme-substrate complex were the rate-determining step. 

In enzyme-catalyzed kinetics, one must necessarily deal with the behavior of a multistep reaction scheme. For initial rate enzyme processes, one typically deals with collections of rate constants which appear in the form of the maximal velocity Um (shortened to U) or the specificity constant VJK (shortened to VIK). Accordingly, enzyme kineticists will use °V and °V/K as an easy way to indicate the respective isotope effects [(Um)H/(Um)D ] and [(VJK )u/(VJK )b], respectively. 

A measure and/or description of how specific an enzyme is toward a substrate or class of substrates or toward an effector or class of effectors. For effectors (or for ligands binding to macromolecules that are not enzymes), this specificity is readily measured by dissociation (or, association) constants. For enzymes, specificity is best quantitated by the Fmax/.K m ratio. See Specificity Constant. It is crucial, in the complete characterization of an enzyme, that the specificity of the enzyme be known in detail. 

With the increased use of enzymes in polymer chemistry, the enzymology terminology to describe the reaction kinetics and the enantioselectivity of a reaction has become more and more common in polymer literature. The parameter of choice to describe the enantioselectivity of an enzyme-catalyzed kinetic resolution is the enantiomeric ratio E. The enantiomeric ratio is defined as the ratio of the specificity constants for the two enantiomers, R) and S) (1) ... 

Since a similar reaction with two stereoisomers leads to different results (one enantiomer reacts, the other does not) kinetic resolution should be called enantiospecific. However, this does not seem to have become common terminology, so it may be better to characterise the reaction by the enantiomeric ratio E. The enantiomeric ratio is the ratio of the specificity constants of the enzyme for the two enantiomers... 

Selectivity is an intrinsic properly of enzymatic catalysis. [3] Following the nomenclature proposed by Cleland [24, 25], the pseudo second-order rate constant for the reaction of a substrate with an enzyme, kml/KM, is known as the specificity constant, ksp. [26] To express the relative rates of competing enzymatic reactions, involving any type of substrates, the ratio of the specificity constants appears to be the parameter of choice [3]. Since the authoritative proposition by Sih and coworkers [27], the ratio of specificity constants for the catalytic conversion of enantiomeric substrates, R and S, is commonly known as the enantiomeric ratio or E -value (Equation 1) ... 

To test the feasibility of enzyme-catalyzed enantiosective reactions in solid/gas reactors and to evaluate the efficiency of the resolution obtained in the gas phase compared to liquid systems, resolution of racemic 2-pentanol, catalyzed by CALB, through alcoholysis with methyl propanoate as acyl donor has been investigated in both liquid media and the gas phase [24]. As CALB has an enantiopreference for R enantiomers of secondary alcohols, this last reaction leads to S-2-Pentanol. This compound is a chiral intermediate in the synthesis of several potential anti-Alzheimer s drugs that inhibit 3-amyloid peptide release and/or its synthesis [25]. The degree of enantioselectivity was measured by using the enantiomeric ratio E, which is defined as the ratio of the specificity constants kcat/KM for the enantiomers (R/S in this case). E can be determined from the enantiomeric excess of... 

The best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio kcat/Km for the two reactions. This parameter, sometimes called the specificity constant, is the rate constant for the conversion of E + S to E + P. When [S] << Km, Equation 6-26 reduces to the form... 

The enzymatic activity of the L-19 IVS ribozyme results from a cycle of transesterification reactions mechanistically similar to self-splicing. Each ribozyme molecule can process about 100 substrate molecules per hour and is not altered in the reaction therefore the intron acts as a catalyst. It follows Michaelis-Menten kinetics, is specific for RNA oligonucleotide substrates, and can be competitively inhibited. The kcat/Km (specificity constant) is 10s m- 1 s lower than that of many enzymes, but the ribozyme accelerates hydrolysis by a factor of 1010 relative to the uncatalyzed reaction. It makes use of substrate orientation, covalent catalysis, and metalion catalysis—strategies used by protein enzymes. 

Volume of first and second vessels Liquid volume Enzyme velocity constant Maximum enzyme velocity constant in unprotonated form Initial enzyme velocity constant Enzyme velocity constant based on unit volume of immobilised biocatalyst Maximum rate of reaction involving substance S Maximum rate of reaction involving substance P Specific rate of generation of biomass fraction Biomass concentration Initial or feed biomass concentration Average biomass concentration Concentration of prey Concentration of predator Biomass concentration at optimum dilution rate... 

Significance of the Specificity Constant, kcat/Km. Under physiological conditions, enzymes usually do not operate at saturating substrate concentrations. More typically, the ratio of the substrate concentration to the Km is in the range of 0.01-1.0. If [S] is much smaller than Km, the denominator of the Briggs-Haldane equation [equation (25)] is approximately equal to Km, so that the velocity of the reaction becomes... 

The ratio kcdt/Km is referred to as the specificity constant. Equation (29) indicates that the specificity constant provides a measure of how rapidly an enzyme can work at low [S]. Table 7.3 gives the values of the specificity constants for some particularly active enzymes. 

The specificity constant kcat/Km is useful for comparing the relative abilities of different compounds to serve as a substrate for the same enzyme. If the concentrations of two substrates are the same, and are small relative to the Km values, the ratio of the rates when the two substrates are present is equal to the ratio of the specificity constants. 

Another use of the specificity constant is for comparing the rate of an enzyme-catalyzed reaction with the rate at which random diffusion brings the enzyme and substrate into contact. We mentioned previously that if every collision between a protein and a small molecule results in a reaction, the maximum value of the second-order rate constant is on the order of 108 to 109m s Some of the values of kcat/Km in table 7.3 are in this range. The reactions... 

Each assay at the indicated substrate concentration was initiated by adding enzyme to a final concentration of 0.01 nM. Derive Km, VmaK, kcat, and the specificity constant. 

Conversely, if [S] < C Km, (Eq. (2.42)) reduces to v = (k2/Km)[E]o[S]. This means that the active sites on the enzyme are effectively unoccupied. The ratio k2/Km is also known as the enzyme s specificity constant, a measure of the enzyme s affinity for different substrates. Thus, if the same enzyme can catalyze the reaction of two substrates, S and S, the relative rates of these two reactions are compared using (k2/Km)s (k2/Km)s-. Because the specificity constant reflects both affinity and catalytic ability, it is also used for comparing different enzymes. 

The E value represents the ratio of the specificity constants of the enzyme for the two enantiomers of the substrate and allows one to compare directly the selectivity of different enzymes in a reaction [15], In contrast, using the purified CLC form of the enzyme gives an E = 66, which makes it a viable method for obtaining pure S-ketoprofen [16]. 

The term cat can be substituted for k2 and is referred to as the turnover number of an enzyme (units of s l). The expression kcJkm is widely used as a measure of the catalytic efficiency of an enzyme and is termed the specificity constant or turnover number. Where [6] Km one can assume that all the enzyme is bound to substrate (i.e., [ 0] = [ES ). Under these conditions the maximal velocity of the reaction Fmax, is a function of... 

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