Kinetics burst

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown 

By means of Eqn. 17 and stopped-flow studies at various values of Sq, k2, and kj, can be separately determined and studied. For carboxypeptidase Y, which also shows such burst kinetics with 4-nitrophenyl trimethylacetate (I), enzyme preparations with differing amounts of attached carbohydrate, reacted with closely similar steady-state parameters K, k ) but differences were apparent for presteady-state parameters (k2 [14]. 

Not only can one obtain rate/mechanistic information from such studies but burst kinetics have provided a method for active-site titration, which determines the concentration of functioning active sites in a suitable enzyme preparation. 

The simplest case is when k2 k and Sq for then w = q, but Eqn. 18 can be applied even when such useful inequalities do not hold. In the studies with carboxypeptidase Y mentioned above [14], the difference in molecular weights of enzyme forms, because of different amounts of carbohydrate attached, was first noticed in burst titration studies and then confirmed using ultracentrifugation, polyacrylamide gels, etc. 

In preliminary studies using 11, which is cleaved by this enzyme to give a fluorescent product, one can easily detect at 0.2 /iM enzyme [15], Active-site titration of isoleucyl-tRNA synthetase, using only 0.1 ml of 1 jiM enzyme has been carried out [16] by means of an aliquot-sampling procedure of radioactive pyrophosphate released as P, in a mechanism formally analogous to that of Eqn. 7. Active-site titration has been reviewed [17]. 

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FIGURE 16.21 Burst kinetics observed iu the chymotrypsiii reaction. A burst of nitrophe-nolate production is followed by a slower, steady-state release. After an initial lag period, acetate release is also observed. This kinetic pattern is consistent with rapid formation of an acyl-enzyme intermediate (and the burst of nitrophenolate). The slower, steady-state release of products corresponds to rate-limiting breakdown of the acyl-enzyme intermediate. 

In the chymotrypsiii mechanism, the nitrophenylacetate combines with the enzyme to form an ES complex. This is followed by a rapid second step in which an acyl-enzyme intermediate is formed, with the acetyl group covalently bound to the very reactive Ser . The nitrophenyl moiety is released as nitrophenolate (Figure 16.22), accounting for the burst of nitrophenolate product. Attack of a water molecule on the acyl-enzyme intermediate yields acetate as the second product in a subsequent, slower step. The enzyme is now free to bind another molecule of nitrophenylacetate, and the nitrophenolate product produced at this point corresponds to the slower, steady-state formation of product in the upper right portion of Figure 16.21. In this mechanism, the release of acetate is the rate-llmitmg step, and accounts for the observation of burst kinetics—the pattern shown in Figure 16.21. 

Kinetics under [substrate] [ligand] Burst Kinetics. . . . 162... 

Fig. 10. Burst kinetics under [substrate] >[ligand-Zn2+ ion complex]...

Figure 10 shows typical examples of burst kinetics observed for the reactions of 29-Zn2+ and 38c-Zn2+ ion complexes under the conditions of excess substrate over ligand. Such burst kinetics can be accounted for a two-step reaction involving an acylated intermediate as in Scheme 4, and the rate constants, ka and kd, can be obtained based on Eqs. 8-11 38,39), where A is the slope of the steady-state line and B is the intercept obtained by extrapolating the steady-state line to time = 0. The ka should be the same with the kc in Table 5. 

Figure 13 indicates burst kinetics. As discussed before, such biphasic curves indicate the reaction to occur through two steps involving an acylated intermediate. The initial slopes for the presteady state can be taken as the measure of acylation rates, and the slopes of the later straight line for steady-state can be taken as the measure of deacylation rates. 

Table 12. Enantioselectivities in the acylation and deacylation steps in the burst kinetics of the reaction of (Z)-Phe-PNP(52)...

These workers examined the mechanism of this reaction in detail (258). Burst kinetics were observed suggestive of the formation of an initial species from the catalyst precursor with subsequent slow turnover. The reaction was found to be pH sensitive, with a break point at pH 7.4, indicating a change in mechanism under these conditions. This pH corresponds to the expected value for secondary Cu(II) alkoxides. Based on this evidence, a formulated mechanism was advanced for this reaction, illustrated in Scheme 29. 

As indicated by FershE , the magnitude of the burst equals [E cr/ve] i /( i + 2 F, which reduces to just the concentration of active enzyme [E cr/vJ if 2 is much smaller than ki. Aside from providing valuable mechanistic information, burst kinetic experiments can be gainfully employed to estimate the fraction of active enzyme i.e., Eactive]l[ totai ) for an enzyme preparation. 

VOLUME STRAIN BUNDLING PROTEIN BUNNETT-OLSEN EQUATIONS COX-YEATS EQUATION ACIDITY FUNCTION BURST KINETICS Buthionine sulfoximine,... 

BURST KINETICS FRAGMENTATION FRANCK-CONDON PRINCIPLE FLUORESCENCE JABLONSKI DIAGRAM FRAP,... 

Several workers have found burst kinetics for various substances at low pH for both Zn(II) and Co(II) enzymes (96, 98, 135-137, 147)- The rate of phosphorylation of the Co(II) enzyme is much faster than the Zn(II) enzyme however, the rate of steady state hydrolysis by the Zn(II) enzyme is twice as fast as by the Co (II) enzyme (96). Burst ... 

Fig. 11.10 (A) Burst kinetics for release of the leaving group from a substrate of a-chymotrypsin. (B) Titration of papain with the irreversible inhibitor 4-toluenesulphonamidomethyl chloromethyl ketone using methyl benzoylglycinate as substrate.

We have pursued such ester hydrolysis by artificial enzymes further. With a cyclodextrin dimer related to 25 we have hydrolyzed an ordinary doubly bound ester, not just the more reactive nitrophenyl esters [116], with catalytic turnovers. Also, with a catalyst consisting of a cyclodextrin linked to a metal ligand carrying a Zn2+ and its bound oxime anion, we saw good catalyzed hydrolysis of bound phenyl esters with what is called burst kinetics (fast acylation, slower deacylation), as is seen with many enzymes [117]. 

The peroxidase reaction (Figure 12.7a) can function in the absence of cyclooxygenase activity, because the intermediate product (prostaglandin G2) can be provided by another enzyme molecule. In accord with this model, a sample of cyclooxygenase, when expressed recombinantly and in the absence of arachidonic acid substrate, will initially be inactive, but it will exhibit burst kinetics upon first contact with arachidonic acid, due to the cascading activation of more and more enzyme molecules by PGG2". ... 

Kunitake and Okahata examined the hydrolysis of PNPA in the presence of vinyl polymers containing N-phenylhydroxamate (PHA) and methylimidazole(MIm) units (107). The reacticm of PHA-MIm-AAm tetpolymer 23 with large excesses of PNPA gives typical burst kinetics initial rapid liberation of p-nitrophenol (k ) followed by slower, steady release (ktt). 

L.K. Nielsen, J. Risbo, T.H. Callisen, and T. Bjorholm. Lag-burst kinetics in phospholipase At hydrolysis of DPPC bilayers visualized by atomic force microscopy. Biochim. Biophys. Acta, 1999, 1420, 266-271. 

Figure 5.38 Burst kinetics showing liberation of aglycone during the establishment of a steady state during hydrolysis of a snbstrate by a retaining glycosidase, when the hydrolysis of the glycosyl-enzyme is rate determining. The glycone was fluorinated, so the experiment was performed in a conventional spectrometer (note time-scale). Note the burst of 0.03 absorbance units.

Burst kinetics are often described in terms of a two-step irreversible mechanism (5, 38). 

The presteady-state burst will be followed by steady-state turnover at a rate given by cat The presteady-state burst of product formation will occur at a rate defined by the sum of the rates of the chemical reaction and product release. The amplitude is also a function of both rate constants, k2 and kj. Thus, the amplitude of the burst can be predicted from the rate of the burst and the rate of steady-state turnover. Although this model can account for burst kinetics, it is often inadequate due to the assumed irreversibility of the chemical reaction. The internal equilibrium arising from the reverse of the chemical reaction k-2) reduces the amplitude of the burst to less than predicted by Eq. (26). 

Because of the factors that reduce the amplitude in a presteady-state burst experiment and the difficulty in resolution of the product (or intermediates) from excess substrate, it is often desirable to use single-turnover methods. These experiments are performed with enzyme in excess over substrate to allow the direct observation of the conversion of substrates to intermediates and products in a single pass of the reactants through the enzymatic pathway. Unlike the presteady-state burst experiments, the kinetics are free of complications resulting from the steady-state formation of products, which limits the resolution of the burst kinetics and the detection of any intermediates above the background of excess substrates and products. 

Fig. 7. Dynein ATPase burst kinetics. The kinetics of a presteady-state burst of ATP binding (o) and hydrolysis ( ) were determined at two ATP concentrations (A) 30 and (B) 50 /xAf. The data fit rate constants of k = 4.7 juAf sec", ki = 55 sec, k-2 = 10 see", and ks = 8 see" according to Scheme IV. Reproduced with permission from (J9).

Presteady-state burst kinetics can be fit to an equation of the form... 

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