Titration of active sites

Non-station ary Methods of Enzyme Kinetics Titration of Active Sites 9.2.5.1 Determination of Concentration of Active Sites... 

TABLE 7 Titration of Active Sites by Various Poisons... 

This condition is useful for the titration of active site concentrations. If Cs(0) is not large enough for the condition Ce(0)=Ces(0+Cep(0 to hold, then the intercept is... 

Figure 11A shows a theoretical example of a titration curve A + B = AB, where the signal is proportional to the amount of complex. The solid lines represent conditions where Bmax is equal to KD. Here for both presentations of signal vs either [Atotal] (total concentration of A added to the preparation) or [Afree] (concentration of non-complexed A in the solution, calculated as [Atotal] - ([AB]) the plot is curved and allows discrimination between free and complexed binding partners. If [Bmax] is substantially higher than KD the issue of active site... 

The weight (or more correctly, the mass) of a protein expressed in grams per mole of active sites. Not all oligomeric proteins, even some with identical subunits, have a number of active sites equal to the number of subunits. Enzyme normality (Le., the concentration of enzyme active sites) is typically determined by active site titration with an active-site-directed irreversible inhibitor. This is... 

The calculation of rate constants from steady state kinetics and the determination of binding stoichiometries requires a knowledge of the concentration of active sites in the enzyme. It is not sufficient to calculate this specific concentration value from the relative molecular mass of the protein and its concentration, since isolated enzymes are not always 100% pure. This problem has been overcome by the introduction of the technique of active-site titration, a combination of steady state and pre-steady state kinetics whereby the concentration of active enzyme is related to an initial burst of product formation. This type of situation occurs when an enzyme-bound intermediate accumulates during the reaction. The first mole of substrate rapidly reacts with the enzyme to form stoichiometric amounts of the enzyme-bound intermediate and product, but then the subsequent reaction is slow since it depends on the slow breakdown of the intermediate to release free enzyme. 

Active site burst titrations of the Zn(II) enzyme and the Co(II) enzyme at acidic pH and low substrate concentrations [(70-1) X 10 6 M] indicated one active site (96, 98, 99, 110, 135), while similar experiments (186) at high substrate concentration (2 X 10-3 M) yielded a value of 2.7 sites per dimer. In the latter experiment, error in the number of active sites might arise from the fact that the results were obtained by extrapolation from steady state measurements without direct observa-ion of the pre-steady-state phase. In the low substrate experiments, the pre-steady-state phase was observed directly. A burst was obtained at intermediate substrate concentrations (4 X 10"4 M), but the size was not reported (137). 

For a-chymotrypsin, the procedure of active-site titration for the calculation of active enzyme concentration and thus of the catalytic constant kcat is long established. The original active-site titration experiment on a-CT by Hartley and Kilby (Hartley, 1954) was performed with ethyl p-nitrobenzoate (Figure 9.2). 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Associated with the problem of active-site titration is the question of the location of the active site in the three-dimensional structure of the protein. As a prelude to this investigation, a study is needed to indicate which amino acid residues in the overall peptide sequence are in the active site. The active site is defined as the location of the enzyme catalysis thus, the substrate complexes at the active site prior to the catalytic process. Addition of a substrate will, therefore, protect the enzyme against reagents, such as inhibitors, which react at the active site. Of course, the active site may include amino acid residues from distant parts of the peptide chain for example, both serine-195 and histidine-57 are in the active site of a-chymo trypsin. 

The notion of turnover numhera was raised by Boudart, but Carberry has emphasised Chat generally we attempt to measure the number of catalyst sites (by chemisorption or titration) rather than the wore relevant number of sites c f catalysis and these are unlikely to be equal. Possibly the alkene titration using reactant cyclohexene would assess the summation of active sites as the active surface area under reaction conditions. An interesClng alternative approach is to use pulsed isothermal alkane titrations Co deduce the numbers of different surface Ptg sites Involved later In hydrogenation. If such a partial deconvolution of total average turnover numbers is really possible, then potentially it Is excemely useful. 

The catalytic work on the zeolites has been carried out using the pulse microreactor technique (4) on the following reactions cracking of cumene, isomerization of 1-butene to 2-butene, polymerization of ethylene, equilibration of hydrogen-deuterium gas, and the ortho-para hydrogen conversion. These reactions were studied as a function of replacement of sodium by ammonium ion and subsequent heat treatment of the material (3). Furthermore, in some cases a surface titration of the catalytic sites was used to determine not only the number of sites but also the activity per site. Measurements at different temperatures permitted the determination of the absolute rate at each temperature with subsequent calculation of the activation energy and the entropy factor. For cumene cracking, the number of active sites was found to be equal to the number of sodium ions replaced in the catalyst synthesis by ammonium ions up to about 50% replacement. This proved that the active sites were either Bronsted or Lewis acid sites or both. Physical defects such as strains in the crystals were thus eliminated and the... 

A stock solution of trypsin (Sigma T-8642 or equivalent) is first standardized using the substrate p-nitrophenol-p -guanidobenzoate HCl (Sigma N-8010). By the initial burst principle (5) one can titrate the active site of trypsin and c culate the uM amount of active trypsin introduced into the rSLPI inhibition assay. By knowing the molecular weight of the trypsin (source dependent) the uM amount of rSLPI present can be deduced from the 1 1 stoichiometry of the reaction. 

While the adsorption of nitric oxide is very useful for the measurement of magnetite surface areas, it is not necessarily true that this molecule titrates the active sites for the water-gas shift reaction. (This point will be discussed later in this paper.) For this reason, recent studies have focused on the adsorptive properties of magnetite for other molecules, and in particular, on the... 

The effect of Si substitution on the turnover frequency for WGS is shown in Figure 11. The turnover frequencies plotted in this figure were based on the magnetite surface area as determined by the NO chemisorption technique. The turnover frequencies shown for unsupported Fe O indicate that the factor of 10 decline in activity for the silica-supported catalysts is not a particle size effect, but instead is a consequence of the substitution of Si into the lattice. However, when the adsorption of CO/COo at 663 K was used to titrate the surface sites instead of NO, the resulting turnover frequencies were essentially constant as shown in Figure 12. Accordingly, the CO/CO2 mixture apparently titrates the sites active for WGS. Clearly, the number of active sites is decreased markedly as the particle size decreases in the silica-substituted magnetite catalysts. 

In closing, it is important to note that the CO/CO2 adsorption technique effectively titrates the active sites for WGS on magnetite catalysts which differ in activity by over an order of magnitude. Nitric oxide on the other hand titrates all of the surface cation sites and is unaffected by Si-substitution. Indeed, NO is known to chemisorb strongly on iron oxides and may even be able to reconstruct the surface. Thus, the combined use of NO and CO/CO2 adsorption provides information about the total magnetite surface area and fraction of the magnetite surface which is active for the WGS reaction. 

Poison titration is a convenient way to measure the concentration of active sites. The best procedure is to use a simple pulse reactor, such as that in Fig. 7.26. Pulses of a poisoning agent are injected between reactant pulses. If all the poison adsorbs irreversibly, then activity declines with each pulse. Typical results arc shown in Fig. 7.27, in which hydrogen sulfide poisons metal sites. Extrapolation of the activity curve to zero gives the amount of poison necessary to neutralize the active sites. A knowledge of surface stoichiometry is necessary to proceed further. For example, in Fig. 7.27 the assumed ratio was two nickel for each sulfur. This technique has the potential for innovative application to many systems. 

DFP titrations of purified serum cholinesterase have indicated two active sites for the horse enzyme (M7) and two active sites for the C4 component of the human enzyme (M20). In other studies using this kind of technique, human serum cholinesterase has been found to have two or three active sites (Y3), and purified horse serum, three or four (M6, T6). A slightly different approach (R9) was adopted by La Du (L34), who titrated the number of active sites of purified usual and atypical human serum enzymes using a carbamyl ester [iV-methyl-(7-dimethyl-carbamoxy)- quinolinium iodide], which is not fluorescent itself but gives a highly fluorescent hydrolysis product. The reaction of the probe (S) with cholinesterase (E) is as follows ... 

Poisons can be used to titrate the active sites many Lewis bases chemisorb strongly onto sites and block polymerization. For example, Hogan [40-42] added triethylamine to a reactor in which polymerization was already occurring on Cr/silica. The polymerization could be retarded or even stopped by the addition of trace amounts of the amine. In one experiment, he injected 0.057 mol of NEt3 per mol of Cr after polymerization had reached a nearly constant rate. The activity immediately dropped by 32%. In a second run, he injected 0.108 mol of NEt3 per mol of Cr,... 

Wu and Lee [166] report that 24 kinds of ion-exchange resin were used to clarify this character of the resin, including six kinds of commercial ion-exchange microresin, five kinds of laboratory-produced macroresin, and 13 kinds of laboratory-produced microresin, using instrumental analysis by TGA, EA, and SEM-EDS, and the reaction method. The densities of active sites in the resin, titrated using the Volhard method for commercial anion exchangers, were higher than those for laboratory-produced resins. 

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