Initial burst enzyme kinetics

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. 

Serine peptidases can hydrolyze both esters and amides, but there are marked differences in the kinetics of hydrolysis of the two types of substrates as monitored in vitro. Thus, the hydrolysis of 4-nitrophenyl acetate by a-chy-motrypsin occurs in two distinct phases [7] [22-24]. When large amounts of enzyme are used, there is an initial rapid burst in the production of 4-nitro-phenol, followed by its formation at a much slower steady-state rate (Fig. 3.7). It was shown that the initial burst of 4-nitrophenol corresponds to the formation of the acyl-enzyme complex (acylation step). The slower steady-state production of 4-nitrophenol corresponds to the hydrolysis of the acetyl-enzyme complex, regenerating the free enzyme. This second step, called deacylation, is much slower than the first, so that it determines the overall rate of ester hydrolysis. The rate of the deacylation step in ester hydrolysis is pH-dependent and can be slowed to such an extent that, at low pH, the acyl-enzyme complex can be isolated. 

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 initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

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". ... 

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... 

A good esterase mimic, exhibiting burst kinetics and turnover, has been constructed by attaching to 3-amino-/3-cyclodextrin a bound zinc cation and an oxime anion. This construct (186) was observed to catalyse the hydrolysis of p-nitrophenyl acetate (PNPA) extremely effectively (A cat/ unoat = 22 600 for the initial fast deacylation step). As in the hydrolysis of PNPA by chymotrypsin, this fast step was followed by a slower reaction in which the acyl- enzyme intermediate suffered hydrolysis.The attachment to the a-face of yS-cyclodextrin of a series of )-aminoalkyl groups has produced a lengthening arm that has permitted a study of the catalysed aminolysis of jo-nitrophenyl acetate (PNPA). The construct containing the three-carbon linker (187 n = 3) was found to be the most effective accelerant, but only two-fold better than that containing the six-carbon linker (187 n = 6). The proposed mechanism involves the fast reversible formation of a complex between the modified CD and PNPA, which, in a slow step, yields the amide (188) (Scheme 34). 

The shapes of the curves in Fig. 6 are consistent with a two-step pathway, analogous to that of a hydrolytic enzyme such as a-chymotrypsin,30 in which an initial acylation burst is followed by a slow deacylation reaction. Following a fast preequilibrium binding, the first kinetic step can be attributed to acylation by substrate of the polymer imidazole residue, accompanied by simultaneous release of nit-rophenol(ate). The succeeding kinetic step would then be ascribed to hydrolysis of the acylimidazole leading to carboxylate ion and regenerated imidazole. 

FIGURE 6-19 Pre-steady state kinetic evidence for an acyl-enzyme intermediate. The hydrolysis of p-nitrophenylacetate by chymotrypsin is measured by release of p-nitrophenoi (a colored product). Initially, the reaction releases a rapid burst of p-nitrophenol nearly stoichiometric with the amount of enzyme present. This reflects the fast acylation phase of the reaction. The subsequent rate is slower, because enzyme turnover is limited by the rate of the slower deacylation phase. 

Under steady-state conditions, the cleavage of this substrate obeys Michaelis-Menten kinetics with a Km of 20 p,M and a fecat of 77 s h The initial phase of the reaction was examined by using the stopped-flow method. This technique permits the rapid mixing of enzyme and substrate and allows almost instantaneous monitoring of the reaction. At the beginning of the reaction, this method revealed a burst phase during which the... 

A positive or negative kinetic cooperativity occurs with a burst" or lag". Burst" is the conformational change of the enzyme (after addition of the substrate) to the form with the lower activity and lag" is the opposite change, in which an increase of the initial speed (rate) is registered. The slow transitions ( bursts" or lags" ) are found at the beginning of the reaction before the steady-state is achieved (9). 

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