Enzyme kinetics turnover number

Horseradish peroxidase is an excellent candidate with which to elucidate enzymatic kinetics in organic solvents. It is an active enzyme with turnover numbers exceeding 320 s 1 in organic media (XI) and hence is susceptible to diffusional limitations which must be overcome. Peroxidase also catalyzes mechanistically identical reactions in aqueous and organic media. Therefore, direct kinetic comparisons between aqueous and organic reactions can be made and the effects of the organic solvent on reactivity and substrate specificity can be directly compared to aqueous-based catalysis. 

The term represents the kinetic efficiency of the enzyme. Table 14.4 lists turnover numbers for some representative enzymes. Catalase has the highest turnover number known each molecule of this enzyme can degrade 40 million molecules of HgOg in one second At the other end of the scale, lysozyme requires 2 seconds to cleave a glycosidic bond in its glycan substrate. 

The question arises as to whether comparisons with protein enzymes are justified. In other words, what can ribozymes really do An important parameter for measuring the efficiency of enzymes is the value of kc-JK. This quotient is derived from the values of two important kinetic parameters kc-Al is a rate constant, also called turnover number, and measures the number of substrate molecules which are converted by one enzyme molecule per unit time (at substrate saturation of the enzyme). Km is the Michaelis-Menten constant it corresponds to the substrate concentration at which the rate of reaction is half its maximum. 

Rhin(bpy)3]3+ and its derivatives are able to reduce selectively NAD+ to 1,4-NADH in aqueous buffer.48-50 It is likely that a rhodium-hydride intermediate, e.g., [Rhni(bpy)2(H20)(H)]2+, acts as a hydride transfer agent in this catalytic process. This system has been coupled internally to the enzymatic reduction of carbonyl compounds using an alcohol dehydrogenase (HLADH) as an NADH-dependent enzyme (Scheme 4). The [Rhin(bpy)3]3+ derivative containing 2,2 -bipyridine-5-sulfonic acid as ligand gave the best results in terms of turnover number (46 turnovers for the metal catalyst, 101 for the cofactor), but was handicapped by slow reaction kinetics, with a maximum of five turnovers per day.50... 

We point out that in enzyme kinetics TON is understood as TOF It is also sometimes called the turnover number, because it is a reciprocal time and defines the number of catalytic cycles (or turnovers ) that the enzyme can undergo in unit time, or the number of molecules of substrate that one molecule of enzyme can convert into products in one unit of time. Quotation from [23]. 

Different from conventional chemical kinetics, the rates in biochemical reactions networks are usually saturable hyperbolic functions. For an increasing substrate concentration, the rate increases only up to a maximal rate Vm, determined by the turnover number fccat = k2 and the total amount of enzyme Ej. The turnover number ca( measures the number of catalytic events per seconds per enzyme, which can be more than 1000 substrate molecules per second for a large number of enzymes. The constant Km is a measure of the affinity of the enzyme for the substrate, and corresponds to the concentration of S at which the reaction rate equals half the maximal rate. For S most active sites are not occupied. For S >> Km, there is an excess of substrate, that is, the active sites of the enzymes are saturated with substrate. The ratio kc.AJ Km is a measure for the efficiency of an enzyme. In the extreme case, almost every collision between substrate and enzyme leads to product formation (low Km, high fccat). In this case the enzyme is limited by diffusion only, with an upper limit of cat /Km 108 — 109M. v 1. The ratio kc.MJKm can be used to test the rapid... 

In the equations describing enzyme kinetics in this chapter, the notation varies a bit from other chapters. Thus v is accepted in the biochemical literature as the symbol for reaction rate while Vmax is used for the maximum rate. Furthermore, for simplification frequently Vmax is truncated to V in complex formulas (see Equations 11.28 and 11.29). Although at first glance inconsistent, these symbols are familiar to students of biochemistry and related areas. The square brackets indicate concentrations. Vmax expresses the upper limit of the rate of the enzyme reaction. It is the product of the rate constant k3, also called the turnover number, and the total enzyme concentration, [E]o. The case u, = Vmax corresponds to complete saturation of all active sites. The other kinetic limit, = (Vmax/KM)[S], corresponds to Km >> [S], in other words Vmax/KM is the first order rate constant found when the substrate concentration approaches zero ... 

A method for deriving enzyme-rate expressions combining both rapid equilibrium and steady-state procedures first illustrated by Chak With this method, demonstrated by Fromm and Huang, a different rate expression will be obtained depending on which steps are chosen to be in rapid equilibrium and which steps are not. See Enzyme Kinetic Derivations Turnover Number S. Cha (1988) J. Biol. Chem. 243, 820. 

Activity Measurements in Solution. The 2 -chloro, 4 -nitrophenyl / -D-glycosides offer an attractive alternative to classical reductometric methods. The substrate is sufficiently stable (pH 5.5, 50°C) and the favorable absorption characteristics of the liberated phenol (pK = 5.5, a/9000 M 1cm 1, pH 5.5 cm 16000 at pH 6.5) allow sensitive, continuous measurements. Kinetic parameters for some of these substrates and enzymes were determined K values were in the mM range for the lactoside (CBH I, EG I, EGD) and were at least 10 times lower for the cellobioside turnover numbers ranged from 1 (CBH I, cellobioside) to 300 min-1 (EG D, cellobioside) (25°C). 

Acetylcholinesterase is subject to substrate Inhibition at high concentrations, but Mlchaells kinetics are observed at lower concentrations, because the substrate constant and the Mlchaells constant differ by a factor of 100. Turnover numbers run about 2-9 x 10 min l, and (Mlchaells constant) values are about 0.2 mM.76,116 Whatever the source, the enzyme is subject to inhibition by the same reversible and irreversible inhibitors. Most of the kinetic work has been done with the saline-extracted 11S enzyme from electric eel and the detergent-extracted 6S enzyme from erythrocytes. The former Is a tetramer derived from the native enzyme by the action of proteases the latter is a dimer. 

A number of cases are known in which the properties of an enzyme are markedly altered by interaction with a membrane. Of course, in some cases the normal function of an enzyme is destroyed when it is removed from the membrane. For example, the mitochondrial coupling factor cannot synthesize ATP when removed from the membrane, since coupling to a proton gradient is required. The portion of the coupling factor that is easily solubilized (F,) is an ATPase. The steady-state kinetic properties of this solubilized ATPase are appreciably changed when it is reconstituted with mitochondrial membranes The turnover numbers and pH dependencies are different the solubilized enzyme is strongly inhibited by ADP, whereas the reconstituted enzyme is not and the reconstituted enzyme is inhibited by oligomycin, whereas the solubilized enzyme is not. 

The parameters kcat and Km also allow us to evaluate the kinetic efficiency of enzymes, but either parameter alone is insufficient for this task. Two enzymes catalyzing different reactions may have the same kcat (turnover number), yet the rates of the uncatalyzed reactions may be different and thus the rate enhancements brought about by the enzymes may differ greatly. Experimentally, the Km for an enzyme tends to be similar to the cellular concentration of its substrate. An enzyme that acts on a substrate present at a very low concentration in the cell usually has a lower Km than an enzyme that acts on a substrate that is more abundant. 

Vmax is important because it leads to the analysis of another kinetic constant, kv turnover number. The analysis of k2 begins with the basic rate law for an enzyme-catalyzed process (Equation E5.5), which is derived from Equation E5.1 ... 

It is also feasible that, following changes in the value of Kmax under different reaction conditions, it might be possible to obtain information concerning the kinetics of the rate-limiting step in the decomposition of ES. The catalytic constant or turnover number ( <, ) is a first-order rate constant that refers to the properties and reactions of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes. The units of kca, are time , and l/k t is the time required to turn over a molecule of substrate on an active site. 

In relation to enzymic cytochrome P-450 oxidations, catalysis by iron porphyrins has inspired many recent studies.659 663 The use of C6F5IO as oxidant and Fe(TDCPP)Cl as catalyst has resulted in a major improvement in both the yields and the turnover numbers of the epoxidation of alkenes. 59 The Michaelis-Menten kinetic rate, the higher reactivity of alkyl-substituted alkenes compared to that of aryl-substituted alkenes, and the strong inhibition by norbornene in competitive epoxidations suggested that the mechanism shown in Scheme 13 is heterolytic and presumably involves the reversible formation of a four-mernbered Fev-oxametallacyclobutane intermediate.660 Picket-fence porphyrin (TPiVPP)FeCl-imidazole, 02 and [H2+colloidal Pt supported on polyvinylpyrrolidone)] act as an artificial P-450 system in the epoxidation of alkenes.663... 

The active-site-directed inhibition of enzymes has been an important research topic in pharmaceutical drug design (Sandler, 1980). An early development of anti-cancer agents involved inhibitions of dihydrofolate reductase and thymidylate synthetase. Search enzyme resource sites for kinetic data (turnover number, Km and Kt) of these two enzymes. 

Initial rates of a hydrolase (10.0 nM)-catalyzed reaction are measured and tabulated overleaf. Evaluate the kinetic parameters of this Uni-substrate reaction. Calculate the turnover number of the enzyme. 

High conversion with an optimal reaction rate [7, 11, 75, 95], increase of the turnover numbers, i.e., the moles of substrate converted per mole of enzyme deactivated [3, 75, 95], and high stereospecificity of the compound of interest are targets of particular interest in the operation of these batch reactors [10,11,48, 77]. The achievement of these goals requires the study of different variables type and concentration of peroxide, substrates and cofactors, enzyme activity and purity, composition of the reaction medium, pH, temperature, or agitation. Such optimization requires a deep knowledge of the system and a mathematical model that represents it satisfactorily. The kinetic model obtained in batch experiments is the... 

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