Molecular activity turnover

Porcine pancreas and porcine pancreatic juice appear to contain a single protein endowed with lipolytic activity. As stated earlier, this protein corresponds to about 2.5 % of the total proteins of the juice. Its molecular activity (turnover number) is likely to be higher than 300,000 under the conditions of the test. Shortage of pure material has thus far prevented any investigation of its molecular properties. It is merely known to be quite soluble in water, to have an isoelectric point of 5.2 in 0.025 M acetate buffer, and to give a conventional protein spectrum. Lipase present in pancreatic juice is likely to be identical with the enzyme extracted from pancreatin. 

Specific activity is defined as the number of enzyme units per unit mass. This mass could correspond to the mass of the pure enzyme, the amount of protein in a particular isolate, or the total mass of the tissue from where the enzyme was derived. Regardless of which case it is, this must be stated clearly. Molecular activity (turnover number), on the other hand. 

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... 

Here Et is the total enzyme, namely, the free enzyme E plus enzyme-substrate complex ES. The equation holds only at substrate saturation, that is, when the substrate concentration is high enough that essentially all of the enzyme has been converted into the intermediate ES. The process is first order in enzyme but is zero order in substrate. The rate constant k is a measure of the speed at which the enzyme operates. When the concentration [E]t is given in moles per liter of active sites (actual molar concentration multiplied by the number of active sites per mole) the constant k is known as the turnover number, the molecular activity, or kcat. The symbol fccat is also used in place of k in Eq. 9-6 for complex rate expressions in which fccat cannot represent a single rate constant but is an algebraic expression that contains a number of different constants. 

The routine unit of enzyme activity has been the international unit (I.U.), namely xmoles P formed (or S consumed) per minute. The specific activity of an enzyme preparation is the number of xmoles P formed (or S consumed) per minute per milligram of protein (clearly this will be very low in a crude cell extract and have a maximal value for a pure preparation of the enzyme). If the molecular mass is known, the specific activity of a pure enzyme measured in saturating (Fmax conditions) can be used to calculate the turnover number (or molecular activity ) of an enzyme, namely the number of P molecules formed (or S molecules transformed) per molecule of enzyme per second (units sec- ). If we recall that the maximal velocity (Fmax) equals k+2 (sec " ) [ET], we can see that the molecular activity equals k+2 (sec -1), that is, fal (sec-1). The katal is the S.I. unit of enzyme activity (moles substrate transformed sec -I) from whence come the corresponding units for specific activity (katal kilogram-1) and molar activity (katal per mole of enzyme). 

The term turnover number can be used in two ways. One way, which has been redefined as molecular activity or molar activity, is the number of moles of substrate transformed per minute per mole of enzyme (units per micromole of enzyme) under optimum conditions. Since many enzymes are oligomers containing n subunits, another possible turnover number is the number of moles of substrate transformed per minute per mole of active subunit or catalytic center (under optimum conditions). This latter definition of catalytic power is called... 

Almost the entire amount of enzyme has been transformed into the ES complex and the active sites of the enzyme are now saturated with substrate. In this case, the coefficient k2 corresponds to the maximum number of substrate molecules that can be transformed into the product by one enzyme molecule (or more exactly, by an active site) per time unit. This number is called turnover number or molecular activity or catalytic constant. Typical values lie between 1 and 10 The name turnover number is unfortunate, though, since 2 represents a frequency (with unit s ) and not a number (unit 1). 

The detection and analysis of O2 evolved are necessary to assure the abihty of the molecular catalyst for water oxidation, for which the activity (turnover rate) and stability (maximum turnover number) of the catalyst should be estimated based on the amount of produced O2. At the same time, attention must be paid to the source of the O atom for O2 evolution in a system including other compounds involving 0 atoms except water. In this section the activity and stability of the catalysts in a solution or in a polymer film, as well as the mechanism for O2 formation including identification of the O atom source, will be reviewed. 

When the molecular weight of a pure enzyme is known it is possible to determine the molecular activity or turnover number,i.e. the number of molecules of substrate transformed per minute per molecule of enzyme. These are usually of the order of several thousand, although acetyl cholinesterase has a value of 950 000 and catalase 5 000 000. 

If the molecular weight of the enzyme is known and if the experiment is carried out with a known quantity of enzyme, the maximal velocity can be expressed in moles of substrate transformed per minute and per mole of enzyme. This value is known as the molecular activity or turnover number. 

Molecular Activity. Many enzymes have thus far been isolated in pure and crystallized form. In these cases, the molecular activity can be determined it is defined as the number of molecules of substrate transformed per minute per molecule of enzyme (or the number of mmoles of substrate per mole of enzyme, i.e. the number of enzyme units p er Mmole of enzyme). The term turnover number" has also been applied to this definition ( nd similar ones). For the calculation of molecular activity, the activity of the enzyme and its molecular weight must be known. A large molecular activity indicates a rapid reaction. Very large molecular activities have been found in the cases of acetylcholinesterase (18 X 10 ). The usual values range from several thousand to ten thousand molecules of substrate per enzyme molecule per minute, still a rather rapid turnover. 

Typical examples are shown in Scheme 6.31. With the catalyst (46), the carbonyl-ene reaction is restricted to both activated aldehydes and reactive 1,1-disub-stituted alkenes. In the presence of molecular sieves, turnover of the catalyst can be realized. 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

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. 

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... 

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-... 

Several reports in which NHC-Pd complexes have been employed to catalyse the copolymerisation of alkenes with CO have appeared over the years. Herrmann and co-workers reported that the chelating dicarbene complex 38 (Fig. 4.14) is active for CO/ethylene [43], The highest TON [(mol ethylene + mol CO) mol Pd ] was 3 075 after a 4 h run. The modest TONs coupled with a very high molecular weight copolymer led the authors to conclude that only a small fraction of the pre-catalyst goes on to form an active species. Low molecular weight (M = 3 790) CO/norbomene copolymer resulted when complex 39 (Fig. 4.14) was tested by Chen and Lin [44]. The catalyst displayed only a very low activity, yielding 330 turnovers after 3 days. 

Figure 3.8. Kinetic data from molecular beam experiments with NO + CO mixtures on a Pd/MgO(100) model catalyst [70]. The upper panel displays raw steady-state C02 production rates from the conversion of Pco = PN0 = 3.75 x 10-8 mbar mixtures as a function of the sample temperature on three catalysts with different average particle size (2.8, 6.9, and 15.6 nm), while the bottom panel displays the effective steady-state NO consumption turnover rates estimated by accounting for the capture of molecules in the support. After this correction, which depends on particle size, the medium-sized particles appear to be the most active for the NO conversion. (Reproduced with permission from Elsevier, Copyright 2000).

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