Enzyme/enzymatic time-dependent

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

Aminocephalosporanic acid (15, Scheme 9) is an important intermediate in the production of many semisynthetic cephalosporin antibiotics (66, 67). However, direct deacylation of cephalosporin C (13) to 15 by cephalosporin C acy-lase is unfavorable, so an enzymatic process is used involving D-amino acid oxidase (DAAO) oxidation of 13 to A-glutaryl-7-aminocephalosporanic acid (14, GL-7-ACA) followed by deacylation to 15 and glutaric acid, catalyzed by GL-7-ACA acylase from Pseudomonas sp. 130 (Scheme 9) (68, 69). GL-7-ACA acylase underwent pseudo first-order time-dependent inactivation by 7 3-bromoacetyl aminocephalos-poranic acid (16) (70). Dialysis did not regenerate enzyme activity, indicating irreversible inhibition. The rate of inactivation was lowered by the presence of either glutaric acid or 15,... 

Enzymes are flexible moieties whose structures exhibit dynamic fluctuations on a wide range of timescales. This inherent mobility of a protein fold was shown to be manifested in the various steps constituting the catalytic cycle. The nature of this linkage between protein structure movement and function undoubtedly is complex and might involve the formation of a coupled network of interactions that bring the substrate closer, orient it properly, and provide a favorable electrostatic environment in which the chemical reaction can occur (45). However, the molecular details that link the catalytic chemistry to key kinetic, electronic, and structural events have remained elusive because of the difficulties associated with probing time-dependent, structure-function aspects of enzymatic reactions. 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

In reversible inhibition, enzymatic activity is regained by the systemic elimination of inhibitor/ such that the time to enzyme recovery is dependent on the elimination half-life of the inhibitor. Competitive inhibition is characterized by competition between substrate and inhibitor for the enzyme s active site. Competition for enzyme binding can be overcome by increasing the concentration of substrate/ thereby sustaining the... 

In order to produce clear juice the cloudy product is treated with pectolytic enzymes to degrade the pectic substances which are responsible for the cloud stability of the cloudy juice. The treatment time depends on the activity of the enzymes and the temperature respectively. Several years ago the industry had no enzymes which were able to work in very acid juice environments (e.g. lemon juice). However, in the meantime new types were developed which are capable of treating acid juices satisfactorily. Some fruit juices contain substances which cannot be removed with pectolytic enzymes and which require other specific enzymatic products to eliminate turbidity, e.g. starch in apple juice requires a suitable amylase. 

The antibiotic chloramphenicol is oxidized by CYP monooxygenase to chloramphenicol oxamyl chloride formed by the oxidation of the dichloromethyl moiety of chloramphenicol followed by elimination of hydrochloric acid " (Figure 33.6). The reactive metabolite reacts with the e-amino group of a lysine residue in CYP and inhibits the enzymatic reaction progressively with time. This type of inhibition is a time-dependent inhibition or a mechanism-based inhibition or inactivation, and the substrate involved historically has been called a suicide substrate because the enzymatic reaction yields a reactive metabolite, which destroys the enzyme. ... 

Finally, enzymatic procedures may be used to produce concentrates of n-3 fatty acids. Depending on the type of enzyme, reaction time, temperature, and the concentration of the reactants and enzyme, it is possible to produce concentrates in different forms, e.g., as free fatty acids or as acylglycerols. Thus, processes such as transesterification, aci-dolysis, alcoholysis, and hydrolysis, as well as esterification of fatty acids with alcohols or glycerol, may be employed. 

The ratio of the rate of the enzymatic reaction to that of diffusion indicates whether the process in an enzyme layer is determined by enzyme kinetics or by substrate diffusion. At low enzyme activity, the process is kinetically controlled. In this case the substrate concentration does not become zero in any part of the enzyme layer, that is, the enzyme sensor signal is mainly a function of the active enzyme concentration. Therefore, effectors (activators, inhibiting factors, including H+, and OH ) and, the amount of enzyme in front of the tranducer, as well as the time-dependent enzyme inactivation, may all directly effect the measuring signal. 

That the inactivation was active-site directed was also established in several ways. As mentioned above, the pA a values of k2 and k, were consistent with the pKa value of catalytic activity for a serine protease. Difference spectra of enzyme with inhibitor showed the reactive product being formed in the presence of enzyme. Rates of inhibition decreased in the presence of a known competitive inhibitor, elastatinal (Okura et al., 1975). The reactive intermediate was generated by mild alkaline hydrolysis and added to assay buffer at a concentration 25 times higher than the Ki of the ynenol lactone. Enzyme and substrate were added to the mixture, and neither inhibition nor time-dependent inactivation was observed. Therefore, inactivation was unlikely to occur by enzymatic release of the reactive intermediate followed by nonspecific alkylation outside the active site. 

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