Catalytic modulation

There are at least three types of PKS. Type I PKSs catalyze the biosynthesis of macrolides such as erythromycin and rapamycin. As modular enzymes, they contain separate catalytic modules for each reaction catalyzed sequentially in the polyketide biosynthetic pathway. Type II PKSs have only a few active sites on separate polypeptides, and the active sites are used iteratively, catalyzing the biosynthesis of bacterial aromatic polyketides. Type III are fungal PKSs they are hybrids of type I and type II PKSs [49,50]. 

As mentioned above the specific structure of HP-RJ has a clear disadvantage for use as universal catalytic module. The substrate binding domain cannot be extended in the 5 direction, requiring HP-RJ to be placed exclusively at the 5 end of a multi-target ribozyme. To overcome this limitation we prepared the branched ribozyme HP-RJBR (Figure 5.2.10). 

With the described set of catalytic modules at hand we designed and synthesized several twin ribozymes, three of those are shown as examples in Figure 5.2.11. 

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1. 

After having shown that combination of two catalytic modules in one molecule results in twin ribozymes with double cleavage activity we wondered whether twin ribozymes can also catalyze RNA ligation and thus be used for catalysis of RNA fragment-exchange reactions. If all fragments (substrates and cleavage products)... 

C0 for a heterogeneous catalyst represents the total concentration ofthe catalytic center or the catalytic module attainable when the catalyst is assumed to be dissolved). 

Adducts Containing Catalytic Modules Synthesized Prior to Incorporation into Polymers... 

To develop a methodology applicable to the design of a wide range of multinuclear active sites on the backbones of insoluble polymers we prepared a molecular entity, composed of various catalytic elements, with a precisely defined structure and then attached it to a polymeric backbone. Thus, we synthesized catalytic modules containing one, two, or four metal-chelating sites, which were subsequently attached to a polystyrene derivative to produce 21-23 [55]. 

Despite of their lack in hydrolytic activity, the CBDs of the cellulases CenA and Cex from C. fimi have been found to be capable of disrupting cotton fibers and releasing small particles from the substrate (Kilburn et al., 1993 Din et al., 1994a). This disruptive effect does not seem to be a general characteristic of CBM, since it was only found with CBD from cellulose from a Penicillium sp. (Gao et al., 2001). Anyhow, a synergism with the catalytic module and enhanced degradation capacity were reported (Din et al., 1994a). 

Parsiegla G, Belaich A, Belaich IP, Haser R. Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases. Biochemistry 2002 41 11134. 

The function of CBM Family 20 (sometimes called SBD, starch binding domains) is particularly well understood in the case where it is attached to a GH 15 catalytic module, as in the Aspergillus spp. glucoamylase. The SBD contains two saccharide binding sites, each of which relies on a Trp residue to interact with the hydrophobic portion of the amylose unit. Mutation of either Trp residue gives an enzyme which continues to bind to soluble starch, but no... 

Peptide synthetases, in general, are large, multifunctional enzymes containing a series of semiautonomous catalytic modules (reviewed by Marahiel et al., 1997 von Dohren et al., 1997). Separate modules for each amino acid (or other carboxy acid) component of a peptide are arranged in series at regular intervals over the length of the polypeptide (Fig. 5). In most eukaryotic peptide synthetases, one large polypeptide contains all the modules and associated activities required to assemble the peptide. However, in several, typically prokaryotic, peptide synthetases, activities are divided between two polypeptides. The substrate specificity of each module from amino to carboxy terminus of the enzyme is co-linear with the structure of the final peptide product. 

Site-Selective Protein Cleavage by Multinuclear Cu(II) Complexes Contained in a Catalytic Module / 108... 

Type I and Type II PKSs catalyze multiple rounds of reactions by catalytic modules encoded either by a single polypeptide (PKS I) or on separate polypeptides (PKS II) by analogy to FAS-I and FAS-II. In contrast, PKS Ills are dimers of KASs that catalyze multiple condensation reactions in one active site and include chalcone synthase, stilbene synthase, and 2-pyrone synthase (see Chapters 1.05, 1.07, and 1.04). In the case of chalcone synthase, three consecutive condensation reactions each utilizing malonyl-CoA, followed by a cyclization reaction, lead to the formation of 4, 2, 4, 6 -tetrahydroxychalcone from 4-hydroxycinnamoyl-CoA (Figure 3). Recruitment of a reductase leads to the formation of a product lacking the 6 -hydroxy group, a reaction that requires an intermediate in the synthesis of chalcone to dissociate from the synthase active site. 

Positive modulation (protection) by substrate has been explained in terms of the stiffness of the enzyme structure that it promotes (Villaume et al. 1990). Negative modulation (destabilization) has also been reported for substrate (Illanes et al. 1998a) and other catalytic modulators (Alvaro et al. 1991). This behavior can be explained in terms of the promotion of quaternary structure dissociation (Misset 1993) and alteration of the oxidation stage in the active site of the enzyme (Bourdillon et al. 1985). 

Fig. 5.21 Simulation of sequential batch reactor operation with immobilised penicillin acylase at pH 7.8 and 40 C (K = 6.5mM Kj = 56.5mM K2 = 59.3 V = 625 pmol/min/g. a) considering full protection by catalytic modulators (all n, = 1 all n( = 1) b) not considering modulation (till n = 0 all nj = 0) c) with experimentally determined vtilues for modulation factors (Illanes et al. 1996)...

The research project allowed to compare two different ways of integrating catalytic reaction and hydrogen separation one in which the membranes are set inside the reaction environment (e.g. the membranes constitute the catalytic tube walls) and another one in which the catalytic module is separated from the hydrogen permeation one. 

The first example published in the literature concerning the use of the hexameric capsule in catalysis was actually an example of catalytic modulation through encapsulation. Scarso and coworkers selected the very well known photocatalyst [Ru(bpy)3] (bpy, 2,2 -fetT-pyridine) that, thanks to its pseudospherical shape and its cationic character, perfectly fits the cavity of the capsule with the aim of investigating the effect of reversible binding on the photocatalytic activity of the metal catalyst [25]. 

Plant pyrones are usually biosynthesized by type III PKSs, which are comparatively simple homodimeric entities (molecular weight usually <50kDa) that work in the iterative mode (the synthase contains a single catalytic module that is used... 

---