Hydrolysis time course

Direct detection of an intermediate. A nice example, the pyridine-catalyzed hydrolysis of acetic anhydride, was discussed in Chapter 1. Spectroscopic techniques are of great value, because they do not perturb the kinetic system, and because they are selective and sensitive. If the intermediate can be detected, the time course of its appearance and disappearance may be followed. 

PentaGalU-ol was used as a substrate for PGI and PGII and triGalU-ol was used for PGX. Substrates were used at 20 mM final concentration in 0.5 ml 100 mM D2O buffer pD 4.5. The amount of enzyme used was such that the rate of hydrolysis was much higher than the rate of mutarotation. Time courses of the reaction mixtures were recorded on a Brucker AM 400 spectrometer at 25 °C. The assignments of relevant resonances was based on data published by Hricovini et al. [5]. 

The cleavage mode of pentaGalU-ol by PGII is essentially the same as found for PGI. Only the secondary hydrolysis reaction of the primary product triGalUA proceeds much more slowly. Spectra are not shown. The time course of the relevant resonances depicted in Fig. 2 demonstrates that the P-anomer of the triGalUA is initially formed. Thus, like PGI, PGII is an inverting enzyme. 

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

Trypsin inhibition The ability of the various trypsin inhibitors Allium extracts) to prevent tiypsin hydrolysis of BAPNA is measured spectrophotometrically (405 nm, = 9.96 cm pmoT ) [11] at 25°C, with a Jasco UV-Vis spectrometer by time course measurement of AAbs. One trypsin unit hydrolyzes 1.0 pmol of A-ft-benzoyl-DL-arginine /i-nitroanilide (BAPNA) per minnte at pH 7.8 and 25°C and one Trypsin Inhibitor Unit (TIU) will decrease the activity of 2 trypsin units by 50%. 

The simple definition of chitinase activity, EC 3.2.1.14, "hydrolysis of iV-acetyl-D-glucosaminide (l-4)-P-linkages in chitin and chitodextrins", belies the complexity and diversity of this group of enzymes. When chitinolytic organisms are investigated in detail, they are found to produce a range of chitinase activities. Usually these can be separated readily by chromatography or electrophoresis. However, it is more difficult to define their precise activities, chiefly because of the uncertain nature of the available assays for chitinases, with non-linear time courses... 

Fig. 5. Time course of the lipase-catalyzed hydrolysis of the (R)- and (5)-ester 1 measured with a UV/ Vis plate reader, (a) WT lipase from P. aeruginosa, (b) improved mutant in the first-generation epPCR 16).

The time course of an actual experiment is shown in Figure 7.17, which shows the hydrolysis of oleic anhydride catalyzed by spontaneously formed oleate vesicles. Note the sigmoid behavior, typical of an autocatalytic process. The lag phase is due to the preliminary formation of vesicles, and in fact the length of the lag phase is shortened when already formed vesicles are pre-added, as shown in the hg-ure. Some mechanistic details of these processes will be discussed in Chapter 10. In this work, an analysis of the number and size distribution of vesicles at the beginning and the end of the reaction was also performed by electron microscopy. 

An enzyme P-galactosidase catalyzes the hydrolysis of a substrate p-nitorophenyl-P-D-glucopyranoside to /i-nilrophenol, the concentrations of which are given at 10, 20, and 40 min in the reaction mixture, as shown in Table P3.6. Table P3.6 Time course of enzyme reaction... 

Consider the hydrolysis of 2,4-dinitrophenyl acetate (DNPA), a compound for which the acid-catalyzed reaction is unimportant at pH > 2 (see Fig. 13.8). In a laboratory class, the time course of the change in concentration of DNPA in homoge-... 

Figure 26.10 Time-course plot of the hydrolysis of flurazepam in 0.1 N H2S04 determined by differential pulse polarography. [From Ref. 131.]...

The ninhydrin reaction (see Basic Protocol 1), the TNBS reaction (see Alternate Protocol 1), the fluorescamine reaction (see Alternate Protocol 2), and formol titration (see Alternate Protocol 3) all evaluate released amino groups by comparing the amounts of free amino groups before and after hydrolysis. The first three methods are spectro-photometric techniques, whereas the fourth is a potentiometric technique. The first and second are chromogenic techniques, whereas the third is fluorometric. These techniques are usually performed as time-course experiments. As the hydrolysis reaction proceeds, aliquots (samples) of the reaction are taken periodically and treated with a test reagent. Products of this reaction are proportional to the amount of free amino groups at each time point. 

The E-3 peak was high in Avicelase activity and in protein content as compared with CMCase activity. This peak was further fractionated on a Bio-gel P-100 column five protein peaks (E-3-1 to E-3-5) were obtained, of which E-3-2 peak was highest among them in Avicelase activity and protein content. The elution patterns are shown in Figure 3, and the time course of hydrolysis of CMC by these cellulase fractions measured by a decrease in the viscosity is shown in Figure 4. Randomness of them is in the order of E-3-5 < E-3-2 < E-3-1 E-3-4 E-3-3. The E-3-2 fraction was subjected to further purification on a CM-Sephadex C-50 column because E-3-5 was very low in the Avicelase activity. 

Evidence for Ex-1 to be an Exo-type Component. The time course of CMC hydrolysis by Ex-1 is shown in Figure 10. The hydrolysis proceeded rapidly at first, but it reached a plateau and seemed to stop after 3 hr. This is characteristic of the hydrolysis by exo-type cellulase, as has been reported for exocellulase of glucosidase type from T. viride (7) and for another Trichoderma exocellulase of Avicelase type (10). 

Similar hydrolysis patterns were observed for the hydrolysis of CMC of different degrees of substitution, and they were entirely different from the patterns obtained by En-1, which was fractionated and purified from the E-4 peak of Figure 1. The purification procedure is not given in this chapter. These time-course patterns are shown in Figures 11 and 12. 

Comparison of Randomness of Ex-1 and Endocellulases on the Hydrolysis of CMC and Cotton. That Ex-1 is least random (as compared with S-l and F-l) on the hydrolysis of CMC and cotton was verified by the observations of the relationships between fluidity of CMC or the decrease in degree of polymerization (DP) of cotton and the simultaneous production of reducing power. These results are shown in Figures 16 and 17. Further, as shown in Figures 18 and 19, the difference in the hydrolysis patterns of both types of cellulase becomes more clear with the comparison between time-course patterns of changes in the viscosity of CMC by both Ex-1 and En-1. The latter is a typical endo-cellulase component as described relative to Figure 12. 

We thus elucidated that three of the four cellulase components are endo- or random-type and the other is exo-type. However, it is difficult to distinguish between the components of least or lowest random-type and those of exo-type. It is rather easy to identify an endo-type cellulase component. In contrast, it is very difficult to determine a cellulase to be exo-type because if the enzyme has a glycosyl-transferring activity the hydrolysis product is not a single sort, which is one of the necessary conditions to be an exo-type. Based on our experiments, measurement of the time course of CMC using a sample of medium substitution degree seems to be the best method of diagnosis to determine a cellulase component to be endo- or exo-type. With some enzymes, direction of mutarotation of reaction products is useful to resolve this problem, as is illustrated by the classic example of the starch hydrolysis by a- and /3-amylases. If this is true for our cellulases, the mutarotation of reaction products would be a... 

Unfortunately, a time course study was not reported in that work, so it is not known whether the crystalline compound corresponds to the -513 or -552 ppm signal of the solution study. However, it seems most likely that the -515 ppm signal corresponds to the bisligand compound of the crystal structure and the -554 ppm signal to the partially hydrolyzed product. Chemically, it seems likely that partial hydrolysis rather than isomerization occurs on dissolution. Additionally, isomerization of VL2 would be expected to provide three NMR signals not just two, whereas two signals would be expected from partial hydrolysis. 

Figure 19. Time course of inactivation of Na+-K+-stimulated ATP hydrolysis ( ) or K+-phosphatase (o) activity of the Na+-K+-ATPase enzyme treated with trypsin or chymotrypsin under carefully controlled conditions in NaCI or KCI media. In NaCI, chymotrypsin (CHY) cleaves at Leu266 (3), while trypsin (TRY) cleaves at Lys30 (2) and Arg262 (3). In KCI, trypsin cleaves at Arg438 (1) and Lys30 (2) in sequence, while there is no cleavage site exposed to chymotrypsin. Data from Jorgensen and Andersen, 1988.

It was found that 46 behaves as an exceptional substrate of trypsin, showing a the reaction mode which had not been observed before. Fig. 3 shows the time course of the tryptic catalysis of 46 monitored by the amidinophenol liberation under the condition that the substrate is in much higher concentration than the enzyme. After rapid mixing of enzyme and substrate, a rapid acylation step is observed and a slow deacylation then follows. The kinetics follow a Michaelis-Menten equation strong binding affinity, efficient acylation, and rate-determining slow deacylation steps, which are exactly the same as those of normal-type substrates. As a result, the accumulation of the acyl enzyme intermediate (EA) is realized in the course of the steady-state hydrolysis [cf. Eq. (6)]. 

Fig. 3. Time course of the trypsin-catalyzed hydrolysis of p-amidinophenyl acetate (46 R = CH3) at pH 8.0, 25 °C. Concentrations of enzyme and ester are 10 pM and 0.7 mM, respectively...

Time-course of the hydrolysis clearly showed in Figure 2 that the reaction proceeded without difficulty even at a substrate concentration as high as 80%, and stopped spontaneously at 50% hydrolysis at which point the acetate of (R)-HMPC was completely hydrolyzed. The HMPC liberated was optically pure at each... 

CH3 to Br to N02. The product / -diketones (largely enolic) were susceptible to hydrolytic retroaldol cleavage, a result which affected the UV spectrum of the reacting solutions. Only when this process occurred at a rate similar to that of the enamine hydrolysis, was the first-order behavior of the time course compromised, and in these cases correction for loss of dione was made. 

The assay was performed by first preincubating selected experimental agents and the enzyme for 10 minutes. Thereafter, the assay was initiated by adding the substrate to obtain a final volume of 100 p,l. The initial velocity of chromogenic substrate hydrolysis was measured by the change in absorbance at 405 nm at 25°C during the linear portion of the time course. Inhibition constants, K, for factor Xa are summarized in Table 1. 

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