Covalent activations

Fig. 2.1L Model of allosteric and covalent activation of glycogen phosphorylase of muscle. The R-form of the subunits are represented by circles, the T-form by squares. The active state of glycogen phosphorylase (GP) is characterized by a high affinity, the inactive state by low affinity for the substrate PI.

Polymerisation carried out in the presence of a coordination catalyst is referred to as coordination polymerisation , when each polymerisation step involves the complexation of the monomer before its enchainment at the active site of the catalyst. The active site in each coordination catalyst comprises the metal atom (Mt), surrounded with ligands, one of which (X) forms a covalent active bond (Mt X) with this metal atom. This implies that the growing polymer chain is covalently bound to the metal atom. A characteristic feature of coordination polymerisation is the mutual activation of the reacting bonds of both the monomer (M) and the active site (Mt-X) through the complexation of the monomer with the metal atom at this site, which results in the cleavage of these bonds in the concerted reaction. 

Previous sections of this chapter have focused on developing general principles for enzyme-catalyzed reactions based on analysis of single-substrate enzyme systems. Yet the majority of biochemical reactions involve multiple substrates and products. With multiple binding steps, competitive and uncompetitive binding interactions, and allosteric and covalent activations and inhibitions possible, the complete set of possible kinetic mechanisms is vast. For extensive treatments on a great number of mechanisms, we point readers to Segel s book [183], Here we review a handful of two-substrate reaction mechanisms, with detailed analysis of the compulsory-order ternary mechanism and a cursory overview of several other mechanisms. 

For the discussion of the reactivity of covalent active species, see Section II.B.6.C.)... 

Thus, bromide-terminated macromolecules are considered covalent active species in polymerization of cyclic imines or oxazolines (cf., Section III.E, F). The same termini would be inactive, however, in the presence of much weaker nucleophiles, e.g., cyclic ethers. Recombination of active species with Br anion would thus result in termination. 

Thus, corresponding macroesters are the covalent active species in the polymerization of cyclic ethers. 

Table 7 Rate Constants of Propagation on Ionic and Covalent Active Species...

It has been found that decarboxylation may be completely eliminated if cationic polymerization of cyclic carbonates is initiated with alkyl iodide or bromide. It is believed that polymerization proceeds with the participation of covalent active species favoring propagation over side reactions leading to C02 elimination [204]. It is interesting to note, that BF3-initiated polymerization of some cyclic carbonates leads to high molecular weight polymers (M > 10s) [205]. 

Finally, an important point which distinguishes covalent activation and allosteric activation should be mentioned. That is their different mode of deactivation. In the first case we have a distinct class of enzymes, the phosphatases, which deactivate and reverse phosphorylation, whereas in the second case deactivation is regulated by dissociation of the activator or binding of inhibitors. 

Davis et al. [89] reported an important work regarding the immobilization of metalloproteins and enzymes on oxidized, purified and vacuum-annealed SWCNTs in aqueous solution. AFM experiments showed that the immobilization is mainly physical, without need for covalent activation or electrostatic interaction. In fact, cytochrome c at pHs below the isoelectric point and ferritin at pHs above the isoelectric point showed an important adsorption obtained just by stirring the nanotubes dispersion (0.03 mg/mL) in dilute protein solutions (50-100 pg/mL) for a given time (2-20 h). GOx could be also adsorbed in a very efficient... 

DEBS appears to have a fairly broad starter unit specificity. Butyryl-CoA and acetyl-CoA are incorporated into the respective Ciq- and Cg-lactones, although at significantly lower yields than propionyl-CoA [160,161] (Fig. 9). Remarkably, DEBS 1 -I- TE can process unreduced and partially reduced intermediates as well. The AT-L in the NH2-terminal part of DEBS 1 covalently activates the starter units with comparable efficiency [161]. In contrast, malonyl CoA is not recognized as an extender unit by acyltransferase domains (R. Pieper, unpublished). When NADPH is excluded from the reaction mixture, a pyran-2-one was synthesized by DEBS 1 -I- TE [ 161 ]. Consistent with this result, incubation of (2S, 3R)-2-methyl-hydroxypentanoyl-NAC thioester and methylmalonyl CoA with DEBS... 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

Intermediates.—Polymerization of vinyl and diene monomers can occur readily via intermediates which carry a fully developed positive or negative charge [see reaction (1)] and in addition there are now a number of well established examples [see reaction (2)] in which propagation takes place substantially via dipolar covalent active centres, where in effect successive monomer molecules... 

Figure 3 Construction of a covalent activity probe for /3-lactamase enzymes was achieved by biotinylation of ampicillin sulfone with EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce)... 

The external ionization involves addition of the monomer molecule to the covalent active species and, thus, means the covalent propagation. 

With respect to the covalent activation in conjugate additions, the catalyst, usually a primary or a secondary amine, can reversibly form a chiral enamine [ 11 ] to activate the nucleophile (D, Fig. 2.2) or a chiral iminium ion [12] to activate the acceptor (E, Fig. 2.2). The detection of enamine intermediates in asymmetric oiganocatalysis has been for a long time the missing piece of evidence for the commonly accepted mechanism of enamine catalysis. This gap has been recently solved with the first detection and structnral characterization of enamine intermediates in proUne-cata-lyzed aldol reactions by real-time NMR spectroscopy [13] and the direct observation of an enamine intermediate in the crystal strnctnre of an aldolase antibody [14]. 

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