Enzyme activation with

Radmacher M, Fritz M, Hansma H G and Hansma P K 1994 Direct observation of enzyme activity with the atomic force microscope Science 265 1577... 

Summarizing the results of many investigations, monosaccharides and such derivatives as D-mannitol and D-glucitol are rather weak acceptors. Disaccharides, including such acceptor products as isomaltose, are much better acceptors, except for certain molecules, for instance leucrose, which is not an acceptor.29,46,47 The decrease of enzyme activity with time has been described in terms of a first-order reaction. The inactivation parameters have been calculated for the immobilized enzyme. The inactivation constants kd were 0.0135 (1/d) when maltose was the acceptor (stabilizing), and 0.029 (1/d) when fructose was the acceptor.38... 

The ability of flavonoids (quercetin and rutin) to react with superoxide has been shown in both aqueous and aprotic media [59,94]. Then, the inhibitory activity of flavonoids in various enzymatic and nonenzymatic superoxide-producing systems has been studied. It was found that flavonoids may inhibit superoxide production by xanthine oxidase by both the scavenging of superoxide and the inhibition of enzyme activity, with the ratio of these two mechanisms depending on the structures of flavonoids (Table 29.4). As seen from Table 29.4, the data obtained by different authors may significantly differ. For example, in recent work [107] it was found that rutin was ineffective in the inhibition of xanthine oxidase that contradicts the previous results [108,109], The origins of such big differences are unknown. 

Diagnosis of lipoprotein lipase deficiency is based on low or absent enzyme activity with normal human plasma or apolipoprotein C-II, a cofactor of the enzyme. 

T. Shimoboji, E. Larenas, T. Fowler, A. S. Hoffman, and P. S. Stayton, Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates. Bioconjugate CherrL 14 (3), 517(2003). 

If an inhibitor has a slow ofif-rate , this may be observed in a continuous assay as a slow recovery in enzyme activity, with an initial rate equivalent to that expected with a reversible inhibitor increasing slowly until the rate is equivalent to that expected with a reversible inhibitor. In the earher example, the rate measured in group 4 would thus be expected to increase slowly almost ninefold, as inhibitor dissociates from the enzyme. 

Figure 3. Mossbauer absorption spectra of aconitase at 4.2 K in zero field. (A) Dithionite reduced enzyme activated with 99.9% enriched Fe. (B) Dithionite reduced enzyme activated with >90% enriched Fe. (C) Apoaconitase incubated with dithionite and Fe. (Reproduced with permission from ref. 39. Copyright 1982 the authors.)...

By analogy to the PDH complex, the a-ketoglutarate dehydrogenase complex is made up of three enzyme activities with a similar array of activities and coenzyme requirements. 

Second, the correlation of change in enzymic activity with the titration of essential sulfhydryl groups has led to a postulation of eight active sites per 480,000 (56). Unfortunately, the possibility of structural changes during such titrations makes interpretation of such data equivocal. However, the observation that urease retained its activity in 8 M urea, where the molecular weight has been reduced at least to 90,000 (7), supports the conclusion above. 

Even with precise measurements, models reflected in Eqs. (5.74) and (5.76) provide a reasonably exact description of the decrease in enzyme activity with time. Deactivation can be characterized in an especially simple manner by the half-life after which 50% of the initial activity remains [Eq. (5.77), with ty2 and kj in hours]. 

Characterization of a mechanism-based inhibitor may involve the estimation of the constants described in section IV, namely, /qnacI, Kh /ccat, and r. The most common approach has been to incubate inhibitor, enzyme, and cofactors together and to determine the decline in enzyme activity with time (26). In practice, this approach often employs the measurement of residual enzyme activity in a subsequent incubation with a specific substrate under conditions that limit further inactivation and competitive inhibition by the inactivator, usually by an appropriate dilution (10-fold or greater) of the original incubate (5). 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

The catalytic mechanism of GSHPx action is very complex and is even now only partially understood. The original 1975 proposal of Ganther[17] is shown in Fig. 4. This mechanism has however been challenged [18] and it is clear that the actual mechanism is very much more complex than that proposed by Ganther[17]. The specificity with respect to substrate is absolute with respect to GSH and even very similar analogues of GSH cannot support the enzymic activity. With respect to the substrate toward which the enzyme catalysis is... 

Numerous ESR studies of the photodamage effect on biologically important molecules have been reported in recent years. Brustad (377) studied the UV-irradiation of the enzyme trypsin and found a linear correlation of the loss of enzyme activity with concentrations of the photoproduced radical. Leterrier and Douzou (378) have applied ESR techniques to study the mechanism of the photoreduction of flabin mononucleotide. A number of investigations deal with photodamage of aliphatic amino acids, glycine peptides, dipeptides, and alanine peptides (379-383). John et al. 

Any enzyme-based analysis consists of measurement of enzyme activity with or without the presence of substances other than substrate. If these substances, when present, modulate the enzyme activity, that is, if they activate or inhibit the activity, then their amounts can be quantified by using classical Michaelis-Menten kinetics. Thus, an enzyme can be used to assay the follovting broad classes of substances substrates, activators, and inhibitors. 

---