Deacylation rate

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. 

Comparing different polyethylenimines (Table III), we observe that the deacylation rate with the lauryl polymer is approximately equal to that for unmodified polymer. Kinetic analysis reveals that the binding of substrate by the respective polymers, as measured by vKd, is not appreciably different, nor is the rate constant k2. With this substrate, then, there is no evidence that added lauryl groups on the polymer increase the effectiveness of the polymer. 

Deacylation rates of acetylsalicylate by isopropylated polymer at pH 7.3 were markedly slower than with unmodified polymer (Table III). With 2.4xlO-2 total residue molar concentration of isopropylated and unmodified poly(ethylenimine), respectively, k2, the pseudo-first-order rate for the former was 1.45 x 10-3 min-1, for the latter 1.2 x 10-1 min-1. Clearly, primary amines are the major sites for aminolysis of aspirin. 

In the earlier kinetic study26 the rate constant for hydrolysis of lauryl-caproylimidazole PEI to caproate ion and laurylimidazole PEI was estimated to be 0.06 min-1 at pH 7.3. In the present study we have found the rate constant for hydrolysis of laurylacetylimidazole PEI to acetate ion and laurylimidazole PEI under similar conditions to be 0.1 to 0.2 min-1. In the earlier study the hydrolysis rate was inferred by an indirect method from the turnover rate in a steady-state situation. In view of the uncertainties in the indirect method and the difference in size of the acyl group in the two cases, the approximate equality of the deacylation rate constants is gratifying. 

When the ester is mixed with the enzyme, there is a rapid exponential phase followed by a linear increase in the absorbance due to the nitrophenol. The rate constant for acylation and the dissociation constant of the enzyme-substrate complex may be calculated from the concentration dependence of the rate constant for the exponential phases (Chapter 4, equation 4.46). (The rate constant of the linear portion gives the deacylation rate, but this is a steady state measurement.) Unfortunately, nitrophenyl esters are often so reactive that the acylation rate is too fast for stopped-flow measurement. 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

The speeding up of the deacylation rate by adding nucleophiles has been used to give the rate constant k2 for deacylation with substrates for which 3 is normally rate-determining. 1,4-Butanediol is a sufficiently good nucleophile that moderate concentrations cause the deacylation rate to become faster than k221 Values of some rate constants obtained by this method are listed in Table 7.3. 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

In addition, as is shown in Table 13, the deacylation which is represented in kd is increased by a factor of more than 103 in poly(PHA-IM-am), compared with poly(PHA-am). As is observed in the deacylation which is catalysed by imidazole moiety in the case of a-chymotrypsin, the catalysis by imidazole moiety in the deacylation of the acylated hydroxamic acid is considered. The turnover number, turnover hi poly(PHA-IM-am) is also high. The deacylation rate of poly(PHA-IM-am)... 

A similar deacylation mechanism is proposed in a micellar catalysis. Lauryl-imidazolylhydroxamic acid in a cationic micelle gives a high acylation rate, deacylation rate and turnover number, which are estimated to be 159 M-1 - sec-1, 4x10 3 sec-1 and 3.2x10 3 sec 1, respectively (88). 

The deacylation rate can be enhanced further by using 4-vinylimida >le unit (PHA-VIm-AAm) 24 (108). The catalytic cycle of the PNPA hydrolysis by this tetpolymer is shown below. 

S—4 shows the pH-rate profile for deacylation. The bifunctional polymers give deddedly hi er (more than one himdied times) deacylation rates. In fact, the rates are greater dian that of acet imidazole (broken line in F 5—4). 

Static interaction with aspartic acid-158. They have accordingly proposed a mechanism in which histidine-159 participates in the catalytic process as a conjugate acid. The pH-dependence of the acylation and deacylation rate constants on an apparent pK near 4 was considered to be caused by a carboxyl group which in its undissociated state led to an inactive conformation of the enzyme. On the other hand, Allen and Lowe (88) argue that the abnormally low pK of histidine-159 can be attributed to its enclosure by a hemisphere of hydrophobic residues, particularly tryptophan-177. A mechanism which portrays histidine as playing a key role in catalysis is presented in Figure 13, although other mechanisms are not necessarily precluded (87, 89). 

The steady-state level of occupancy of the active site, which is dependent on the ratio of deacylation rate to the acylation rate (fcdeacyi/ acyi) [14], was greater than 0.999. The low deacylation rate was reflected in a very low net hydrolysis rate (Fig. 1), with 1 M- s... 

Firstly, not all enzymes exhibit activity in the crystal (e.g., lysozyme) because neighbouring molecules in the crystal lattice block access to the active site. Secondly, in those enzymes where a conformational change is an obligatory part of the reaction, a reduction in rate may be anticipated if these conformational changes cannot be accomplished readily in the crystal. Thirdly, there is a limitation imposed by diffusion of substrate into and products out of the crystal. Quiocho and Richards [167] showed that with carboxypeptidase Aj, crystals of 5 pm or less were required before the specific activity of the enzyme in the crystal became independent of crystal size. Rossi and Bernhard [168,169] studied the deacylation rate of co-crystallised acylated a-chymotrypsin using a chromophoric substrate. Under conditions where diffusion away of product was not essential for detection of reaction, they showed that deacylation rates were the same in the crystal as in solution. 

Figure 2 Schematic representation of acylation of serine proteases by amidinophenyl benzoates deacylation rate (ks) depends on benzoic acid substitution (R).

To demonstrate the effect of the active-site acylated enzyme, we prepared the benzoyl derivatives of bovine thrombin [22,23]. If benzoyl-thrombin is added to blood plasma, the clotting process is retarded in accordance with the deacylation rate (Fig. 7). 

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