Active-site titration

Titration of the intact active site obviates problems due to inactive protein which contribute to a false molarity. Active-site titrations of acyl group transfer enzymes such as a-chymotrypsin utilise a substrate which has a good leaving group. This enables the buildup of an acyl enzyme intermediate which forms faster than it can degrade and results in 

In Equation 11.13, A = k2k3[E]0[S]0/(k2 + k3) / [S]0 + k3Ks/(k2 + k3), which has the form of a Michaelis-Menten equation, B = [E]0[S]0 /(fe + 3) 2/([S]0+ Km(apparent)), and b is a composite rate constant describing the build-up of the acyl enzme intermediate (or, in the general case, the covalently bound enzyme intermediate). The non-linear plot of [Lg ] against time is shown in Fig. 11.10A for a typical substrate of a-chymotrypsin extrapolation of the linear portion gives the intercept shown which allows evaluation of B. 

When [Lg ] can be measured directly, it is clearly ideal if k3 is relatively small and less than k2, for such reactions, measurements can be made before the overall reaction is complete. Also, it is necessary to be able to measure k2 in order that the equation for B can be analysed. An ideal case would be a reaction where k2 = 0 here, the substrate would essentially be an irreversible inhibitor and the value of B becomes [E]0. 

If it is not possible to measure [Lg ] explicitly by analytical measurements, an irreversible inhibitor can be employed in conjunction with a rate assay using a substrate. Increasing 

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Fig. 11. A Theoretical binding curve for an A + B = AB interaction. Black lines indicate a plot of signal vs [Atotal], Grey lines vs [Afree]. The dashed line shows an example of an active site titration. B Scatchard plot [154]...

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

Selected entries from Methods in Enzymology [vol, page(s)] Active site titration [by 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone, 19, 6-14 by p-nitrophenyl ester substrates, 19, 14-20 by rapidly reversible, covalently bound substrates, 19, 14-20 by slowly reversible, covalently bound inhibitors, 19, 6-14] assay,... 

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... 

AFFINITY ABELING ACTIVE-SITE TITRATION ACETYLCHOLINESTERASE AFFINITY ABELING DIISOPROPYLFLUOROPHOSPHATE ACTIVE TRANSPORT ION PUMPS... 

ACYL ENZYME INTERMEDIATES ACTIVE SITE TITRATION SERPINS (INHIBITORY MECHANISM)... 

BOROHYDRIDE REDUCTION ACYL-SERINE INTERMEDIATE ACTIVE SITE TITRATION Acyl transfer from a peptidyl-tRNA,... 

Even though, the immobilization procedure should be engineered to maximum retained activity of immobilized enzyme, it is difficult to measure the amount of active enzyme on the carrier without an active site titration. However, this has been done in the case of immobilized trypsin, although only covalent immobilized. (Daly and Shih, 1982)... 

Active-site titration versus rate assay... 

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. 

The accumulation of E-Tyr-AMP in the absence of tRNA and the stability of the complex were crucial for the initial success of protein engineering. These factors allow active-site titration and pre- steady state kinetics. Further, the longterm stability of E Tyr-AMP enables the direct solution of its structure by x-ray crystallography. 

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). 

Meanwhile, reagents (and methods see Hsia, 1996) have been improved. A short series of titration experiments at different concentrations of electrophile should be conducted to determine the percentage of active enzyme (without nucleophile, so as not to start the reaction). The calculation of the true [E] (= [E] ) is performed with [El after weighing in, with the factor ffrom the active-site titration experiment from Eq. (9.10). 

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. 

C. Y. Hsia, G. Gantshaw, C. Paech and C. J. Murray, Active-Site Titration of Serine Proteases Using a Fluoride Ion Selective Electrode and Sulfonyl Fluoride Inhibitors, Anal. Biochem. 1996, 242, 221-227. 

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. 

R Kittelberger, M Pavela-Vrancic, El von Dohren. Active site titration of gramicidin S synthetase 2 evidence for misactivation and editing in nonribosomal peptide biosynthesis. FEBS Lett 461 145-148, 1999. 

Once the hexameric structure of the yeast FAS was established, the number of functional active sites still remained to be determined. Earlier studies had shown that the functional complex contains approximately six equivalents each of two prosthetic groups 4 -phosphopantetheine [60,63], necessary for the AGP functionality, and flavin mononucleotide [64], an essential component of the enoyl reductase activity. These studies provided an early indication that each of the six active sites in the complex has a full set of the chemical groups necessary for fatty acid synthesis. Nevertheless, conflicting reports appeared in the literature as to the competence of six active sites. Whereas some reports suggested the possibility of half-sites reactivity (only three of the six sites are catalytically competent) [65, 66], others proposed that all six active sites could synthesize fatty acids [62]. Subsequent active site titration experiments were performed which quantitated the amount of fatty acyl products formed in the absence of turnover [67]. Single-turnover conditions were achieved through the use of... 

Although the above experiments established the dimeric structure of the animal FASs, further work was necessary to establish that each of the two active sites is competent for the synthesis of fatty acids. Active site titrations, performed by inhibiting the thioesterase domain of the synthase and quantitating the bound fatty acyl products that accumulate as a result, indicated that 1 mole of fatty acyl product is formed for each mole of phosphopantetheine present [82]. Thus, each of the two subunits is chemically competent to perform all the necessary reactions of fatty acid synthesis. 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

Active-site Titration. In investigating Immnhl1 -Igod enzymes. 

X 10 moles trypsin per liter fluid volume. To demonstrate the feasibility of using the Ford method to determine the active-site of our immobilized enzyme systems, trypsin CVB-PHEMA-PABS-carbamate was treated in a circulation reactor with NPGB and the titration is Illustrated in Figure 4. The amount of p-nitro-phenol produced by the burst is equal to the amount of the active immobilized trypsin which, for this particular system, turns out to be 31% of the total bound enzyme. Active-site titrations of soluble trypsin were performed according to Chase and Shaw (16), and the active molecules for free trypsin was found to be 70% of the total protein involved. Consequently, the retention of active molecules for the immobilized enzyme was calculated 45%. The specific activity is 17% (Table III) for the same system so the efficiency of the system, based on the actually available active sites, was 38%. Thus, 62% of the initially active trypsin bound has lost its activity upon binding. 

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