Turnover numbers activation

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Carbamate Insecticides. These are stmcturaUy optimi2ed derivatives of the unique plant alkaloid physostigmine [57-47-6] a cholinergic dmg isolated in 1864 from Phjsostigma venenosum (see Alkaloids) (17,24,35—39). The carbamates maybe considered synthetic derivatives of the synaptic neurotransmitter acetylcholine, with very low turnover numbers. The A/,A/-dimethylcarbamates of heterocycHc enols (36) and the Ai-methylcarbamates of a variety of substituted phenols (35) with a wide range of insecticidal activity were described in 1954 (35). The latter are the most widely used carbamate insecticides, and the A/-methylcatbamates of oximes have subsequentiy been found to be effective systemic insecticides. 

The data most frequentiy collected and reported in catalyst performance evaluations are activity or turnover number, selectivity to the desired product(s), overall yield, catalyst life, and the identities and yields of by-products produced. These data are used to further catalyst or process development research efforts, to monitor catalyst manufacture, and to provide quaUty assurance information to catalyst users. 

Enzyme and substrate first reversibly combine to give an enzyme-substrate (ES) complex. Chemical processes then occur in a second step with a rate constant called kcat, or the turnover number, which is the maximum number of substrate molecules converted to product per active site of the enzyme per unit time. The kcat is, therefore, a rate constant that refers to the properties and reactions of the ES complex. For simple reactions kcat is the rate constant for the chemical conversion of the ES complex to free enzyme and products. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

The turnover number of an enzyme, is a measure of its maximal catalytic activity, is defined as the number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate. The turnover number is also referred to as the molecular activity of the enzyme. For the simple Michaelis-Menten reaction (14.9) under conditions of initial velocity measurements, Provided the concentration of... 

Szent-Gyorgyi further showed that the viscosity of an actomyosin solution was lowered by the addition of ATP, indicating that ATP decreases myosin s affinity for actin. Kinetic studies demonstrated that myosin ATPase activity was increased substantially by actin. (For this reason, Szent-Gyorgyi gave the name actin to the thin filament protein.) The ATPase turnover number of pure myosin is 0.05/sec. In the presence of actin, however, the turnover number increases to about 10/sec, a number more like that of intact muscle fibers. 

Quantum yield and luciferase activity The quantum yield of coelenterazine in the luminescence reaction catalyzed by Oplophorus luciferase was 0.34 when measured in 15 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl at 22°C (Shimomura et al., 1978). The specific activity of pure luciferase in the presence of a large excess of coelenterazine (0.9pg/ml) in the same buffer at 23°C was 1.75 x 1015 photons s 1 mg-1 (Shimomura et al., 1978). Based on these data and the molecular weight of luciferase (106,000), the turnover number of luciferase is calculated at 55/min. 

Renilla luciferase. The luciferase of Renilla reniformis has been purified and characterized by Karkhanis and Cormier (1971) and Matthews et al. (1977a). The purified luciferase has a molecular weight of 35,000, and catalyzes the luminescence reaction of coelenterazine. The luciferase-catalyzed luminescence is optimum at pH 7.4, at a temperature of 32°C, and in the presence of 0.5 M salt (such as NaCl or KC1). The luciferase has a specific activity of 1.8 x 1015 photons s"1mg"1, and a turnover number of 111/min. The luminescence spectrum shows a maximum at 480 nm. The absorbance A28O of a 0.1% luciferase solution is 2.1. The luciferase has a tendency to self-aggregate, forming higher molecular weight species of lower luminescence activities. 

When a small fraction of irreversible mediator side reactions cause a rapid decrease of catalytic activity in the homogeneous case in a modified electrode this would be disastrous since there is no bulk supply of catalyst. Thus, higher turnover numbers are generally required than in the homogeneous case... 

Theories neglect that catalysts usually have limited turnover numbers due to destructive side reactions. This may not be so obvious in analytical experiments but it has severe consequences for large scale applications. A simple calculation can illustrate this problem if a redox polymer with a monomer molecular weight of 400 Da and a density of 1 g cm " is considered with all redox centers addressable from the electrode and accessible to the substrate with a turnover number of 1000, then, to react 1 nunol of substrate at a 1 cm electrode surface, at least 5 pmol of active catalyst centers corresponding to 2 mg of polymer, or a dry film thickness of 20 pm are required. This is 20 times more than the calculated optimum film thickness for rather favorable conditions... 

As discussed before, very high turnover numbers of the catalytic site and a large active electrode area are the most important features for effective catalysis. In the following sections three relatively successful approaches are illustrated in detail, all of which make use of one or both of these parameters. A further section will deal with non-redox modified electrodes for selectivity enhancement of follow-up reactions. 

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. 

The oxidation of phenol, ortho/meta cresols and tyrosine with Oj over copper acetate-based catalysts at 298 K is shown in Table 3 [7]. In all the cases, the main product was the ortho hydroxylated diphenol product (and the corresponding orthoquinones). Again, the catalytic efficiency (turnover numbers) of the copper atoms are higher in the encapsulated state compared to that in the "neat" copper acetate. From a linear correlation observed [7] between the concentration of the copper acetate dimers in the molecular sieves (from ESR spectroscopic data) and the conversion of various phenols (Fig. 5), we had postulated [8] that dimeric copper atoms are the active sites in the activation of dioxygen in zeolite catalysts containing encapsulated copper acetate complexes. The high substratespecificity (for mono-... 

Attempts to perform hydrodefluorination on other polyfluoroarenes using 9c met with mixed success (Table 8.4). Pentafluoropyridine proved to be highly active with a turnover number of 13.6, although the distribution of products revealed up to three hydrodefluorination reactions (Table 8.4, entry 4). Substrates with a lower fluorine content, such as CF.CH, and 1,2- or 1,4-CF,H were unreactive. 

They have an exceedingly high specific activity per active site the turnover number y is as high as 10 to 10 s in certain enzyme reactions, while at ordinary electrocatalysts having a number of reaction sites on the order of 10 cm , yhas a value of about 1 s at a current density of lOmA/cm. Thus, the specific catalytic activity of tfie active sites of enzymes is many orders of magnitude fiigher tfian tfiat of all other known catalysts for electrochemical (and also chemical) processes. 

Sterically demanding, water-soluble alkylphosphines 6.10 and 6.11 as ligands have been found to have a high activity for the Suzuki coupling of aryl bromides in aqueous solvents (Eq. 6.35).115 Turnover numbers up to 734,000 mmol/mmol Pd have been achieved under such conditions. Glucosamine-based phosphines were found to be efficient ligands for Suzuki cross-coupling reactions in water.116... 

Different enzymes exhibit different specific activities and turnover numbers. The specific activity is a measure of enzyme purity and is defined as the number of enzyme units per milligram of protein. During the purification of an enzyme, the specific activity increases, and it reaches its maximum when the enzyme is in the pure state. The turnover number of an enzyme is the maximal number of moles of substrate hydrolyzed per mole of enzyme per unit time [63], For example, carbonic anhydrase, found in red blood cells, is a very active enzyme with a turnover number of 36 X 106/min per enzyme molecule. It catalyzes a very important reaction of reversible hydration of dissolved carbon dioxide in blood to form carbonic acid [57, p. 220],... 

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