Modified Enzymes

Modified enzymes can be created by leaving their protein scaffold basically intact but altering some of their properties by chemical or genetic methods, which yields altered and/or improved enzymes for synthetic purposes. The following general strategies are discussed below [457]  

Site-directed chemical modification of enzymes using group-specific reagents was established mainly during the 1960s aiming at the elucidation of enzyme strucmres and mechanisms [458,459] rather than for the creation of biocatalysts with a better performance. In other words, enzyme modification has been developed more as 

Surface-Modified Enzymes. Enzymes acting in nearly anhydrous organic solvents always give rise to heterogeneous systems (Sect. 3.1). In order to turn them into homogeneous systems, which can be controlled more easily, proteins can be modified in order to make them soluble in lipophilic organic solvents. This can be readily achieved by covalent attachment of the amphipathic polymer polyethylene glycol (PEG) onto the surface of enzymes [460]. The pros and cons of PEG-modified enzymes are as follows [461, 462]  

The cyanuric chloride/PEG method seems to work for all classes of enzymes, including hydrolases (lipases [469], proteases, glucosidases [470]) and redox enzymes (dehydrogenases, oxidases [471]). The residual activities are usually high (50-80%), and for most enzymes about five to ten PEG chains per enzyme molecule are sufficient to render them soluble in organic solvents. Care has to be taken to avoid extensive modification which leads to deactivation. PEG-modified enzymes may be recovered from a toluene solution by precipitation upon the addition of a hydrocarbon such as petroleum ether or hexane [472]. 

The majority of applications using PEG-modilied enzymes have involved the synthesis of esters [146, 474 78], polyesters [479], amides [480], and peptides 

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Fig. 8. Entrapment of mediator-modified enzymes within a conductive polymer film where ( ) represents the mediator ferrocene and (B) the active site...

Enzymes. Protein engineering has been used both to understand enzyme mechanism and to selectively modify enzyme function (4,5,62—67). Much as in protein stabiUty studies, the role of a particular amino acid can be assessed by replacement of a residue incapable of performing the same function. An understanding of how the enzyme catalyzes a given reaction provides the basis for manipulating the activity or specificity. 

Anistreplase has a considerably longer a half-life than streptokinase, ie, 90 min compared to 20 min (87,88). Moreover, it does not require prolonged infusion to achieve its thrombolytic effects. Anistreplase was found to be highly effective after a single intravenous dose of 30 units over a 5-min period compared to a 60-min infusion of 1.5 million units of streptokinase (89—94). In direct comparative studies, anistreplase was as effective as intracoronary (95,96) and intravenously (96—100) adrninistered streptokinase. In a randomized, double-blind, placebo-controUed study (AIMS trial) with 1004 patients given this modified enzyme, the 30-day mortaUty rate was 12.2% for patients receiving placebo, compared to 6.4% for patients who received 30 units of anistreplase intravenously within six hours of the onset of symptoms (101). 

Poly(ethylene glycol) (PEG) molecules attached to adenosine deaminase (ADA) have been used in patients exhibiting symptoms of the severe combined immunodeficiency syndrome (SCID) caused by ADA deficiency. The modified enzyme has a plasma half-life of weeks as compared to the unmodified enzyme (minutes) (248). PEG-L-asparaginase has induced remissions in patients with non-Hodgkin s lymphoma (248). However, one disadvantage of PEG-enzyme treatment is its expense, ie, a year s treatment costs about 60,000 (248). 

Reactions that fit this model are called ping-pong or double-displacement reactions. Two distinctive features of this mechanism are the obligatory formation of a modified enzyme intermediate, E, and the pattern of parallel lines obtained in double-reciprocal plots (Figure 14.19). 

The overall direction of the reaction will be determined by the relative concentrations of ATP, ADP, Cr, and CrP and the equilibrium constant for the reaction. The enzyme can be considered to have two sites for substrate (or product) binding an adenine nucleotide site, where ATP or ADP binds, and a creatine site, where Cr or CrP is bound. In such a mechanism, ATP and ADP compete for binding at their unique site, while Cr and CrP compete at the specific Cr-, CrP-binding site. Note that no modified enzyme form (E ), such as an E-PO4 intermediate, appears here. The reaction is characterized by rapid and reversible binary ES complex formation, followed by addition of the remaining substrate, and the rate-determining reaction taking place within the ternary complex. 

Note that these schemes predict that A and Q compete for the free enzyme form, E, while B and P compete for the modified enzyme form, E. A and Q do not bind to E, nor do B and P combine with E. 

Shaw KJ, Rather PN, Hare RS et al (1993) Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Mol Biol Rev 57 138-163... 

Like HATs, most functional HDACs are embedded in large multifunctional protein complexes, which also contain other chromatin modifying enzymes and coregulator proteins [1]. 

Microbial Resistance to Drugs. Table 2 Antibiotic modifying enzymes [2, 3]... 

In the case of liganded NRs, ligand binding is the first and ciucial molecular event that switches the function of these transcription factors from inactive to active state by inducing a conformational change in the LBD of the receptor (Fig. 1). This specific conformation allows the second step of NR activation that corresponds to the recruitment of coregulatoiy complexes, which contain chromatin-modifying enzymes required for transcription. The transcriptional coactivators are very diverse and have expanded to more than hundred in number. These include the pi 60 family of proteins,... 

Marban C, Suzanne S, Dequiedt F, de Walque S, Redel L, Van Lint C, Aunis D, Rohr O (2007) Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J 26(2) 412-423... 

AGAC-modifying enzymes are active outside the cytoplasmic membrane, in the periplasmic space in Gram-negative bacteria and extracellularly in Gram-positives. Table 9.4 summarizes some of the enzymes involved in AGAC resistance and their spectrum of activity. 

Table 9.4 Examples of aminoglycoside-aminocyclitol susceptibility to modifying enzymes... 

Bacterial resistance to anhbiotics is often achieved by the eonstitutive possession or indncibility of dmg-inactivating or -modifying enzymes. This problem ean, at least to some extent, be overcome by designing new dmgs that ... 

Not a great deal is known about factors that actually activate tryptophan hydroxylase. In particular, the relative contribution of tryptophan supply versus factors that specifically modify enzyme activity under normal dietary conditions is unknown. However, removal of end-product inhibition of tryptophan hydroxylase has been firmly ruled out. Also, it has been established that this enzyme is activated by electrical stimulation of brain slices, even in the absence of any change in tryptophan concentration, and so other mechanisms are clearly involved. 

Reaction of purified Ca " -ATPase with 0.3 mM NBD-Cl in the presence of 1 mM AMP-PNP and 1 mM CaCl2 caused inhibition of ATPase activity with the incorporation of 2= 15 nmol NBD-Cl per mg protein [335]. The inhibition was attributed to the binding of 7-8 nmol NBD-Cl/mg enzyme protein, corresponding to = 1 mol NBD-Cl per mol ATPase. The NBD-labeled enzyme was digested with pepsin and several NBD-labeled peptides were isolated [335]. All peptides contained the Gly-X (Cys) sequence that occurs only in one place in the Ca -ATPase, i.e., at Gly343-Cys344. Therefore NBD-Cl reacts with the same cysteine 344 residue that is also modified by maleimide derivatives [319]. The NBD modified enzyme had only 5-10% of the ATPase activity of the control ATPase, but the steady state concentration of the phosphoenzyme intermediate was only slightly reduced [335]. The Ca ... 

These data demonstrate that both GSH and GSSG have profound effects on Na/K ATPase activity and may act in concert to modify enzyme activity during oxidant stress. However, it should be recognized that the steric conformation of an isolated enzyme preparation in a chemically buffered solution may be considerably different to the native enzyme located in a dynamic lipid bilayer. For this reason, these investigations have been extended to include a variety of preparations in which the Na/K pump is in its native environment. 

Acid-catalyzed matrices, kinetics of controlled release, 170-179 Active targeting, definition, 276 Adenosine deaminase, activity of polyethylene glycol modified enzymes, 98-99 Adjuvax... 

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