Enzyme donor specificity

Acyl transfer to alcohols and amines is related mechanistically to ester hydrolysis but yields a complementary set of products that are useful in their own right and as chiral synthons for the preparation of more complex materials. Such transformations can be difficult to achieve in water, however, because the solvent, which is present in vast excess, can participate directly in the reaction as a reactant. Enzyme-like specificity is thus required to favor the bimolecular reaction between alcohol and ester and prevent spontaneous hydrolysis of the acyl donor. 

Modifications of the Sialyl Donor. One of the first reports on the donor specificities of nonnatural substrates described transfer studies using MUNeuSAc and derivatives, with lactose as acceptor, and the native enzyme.166 Derivatives of C-7 MUNeuSAc... 

The [3-galactosidase analog consists of two subunits a large dimeric polypeptide (200 kDa), denoted enzyme acceptor (EA)2, and a small polypeptide (20 kDa), denoted enzyme donor (ED). (EA)2 and ED are enzymatically inactive but spontaneously associate to give enzymatically active tetramers. In CEDIA assays, [45,46] the hapten or analyte is covalently linked to the ED, and an analyte-specific antibody is used to inhibit the assembly of the enzymatically active tetramers. Analyte in a patient s serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of active B-... 

There have been more than 20 aldolases isolated, eight of which have been explored for organic synthesis (6). Aldolases possess two interesting common features the enzymes are specific for the donor substrate but flexible for the acceptor component, and the stereochemistry of aldol reaction is controlled by the enzyme not by the substrates. In our previous study, we have described the use of lipases, hexokinases, glycosyl transferases and rabbit muscle aldolase for the synthesis of certain fluorosugars (7). This review describes our recent development in aldolase-catalyzed reactions for the synthesis of fluorosugars. 

To date commercial /3(l-4)ga/actosyl transferase from bovine milk - /J(l-4)GalT -is the most thoroughly studied transferase with respect to its scope of the acceptor- and donor-specificities [28, 48]. The enzyme transfers a D-galactose unit from UDP-Gal onto the 4-OH-group of a terminal N-acetylglucosamine acceptor in a /1-mode to form N-acetyllactosamine (Fig. 3). In the presence of a-lactalbumine, the preferred acceptor is glucose to give lactose, respectively. 

P-Enzyme is specific for a-n-glucosyl phosphate as donor, but an effective acceptor (primer) must contain a linear chain of at least four... 

In the transaminase reaction, the L-amino acid is the most common donor, but D-amino acids have also been described in that role. Studies on the specificity of transaminase suggest that the enzyme is specific for both the donor of the amino group and the acceptor keto acid. Transaminase requires pyridoxal phosphate as coenzyme for activity (the role of the pyridoxal phosphate in the reaction is discussed further in the section on vitamins). 

It has been purified from wheat flour in which its activity is relatively high (Table 15.22). The enzyme is specific for the H-donor (Table 15.23) because it oxidizes only GSH, and with a much lower velocity also y-glutamyl cysteine, but neither cysteinyl glycine nor cysteine, which also occur in wheat flour (Table 15.24). The specificity for the H-acceptor is not so pronounced. As shown in Table 15.23, all four diastereomeric forms of dehydroascorbic acid are converted, but with different velocities. The substrate specificity corresponds to the varying activity of... 

The particular amino acid side chains (a-substituents) at the active site ate responsible for the enzyme s specificity. For example, an amino acid with a negatively charged side chain can associate with a positively charged group on the substrate, an amino acid side chain with a hydrogen-bond donor can associate with a hydrogen-bond acceptor on the substrate, and a hydrophobic amino acid side chain can associate with hydrophobic groups on the substrate. 

The occurrence of iV -succinyl-a-amino- -ketopimelio acid and its transamination to succinyl-L-diaminopimelic acid has also been reported 196). The transaminase has been purified about 150-fold chiefly by means of chromatography on DEAE-cellulose. This transaminase was shown to be distinct from several other transaminases present in E. coli. The purified enzyme was specific for glutamate as an amino group donor in the formation of succinyldiaminopimelic acid. Succinyl-meso-diaminopimelic acid was inactive as a substrate. 

Norlaudanosoline is metabolically methylated in the 6-position first. Hence it follows that a second 0-methyl group has to be introduced at position 4 of the tetra-hydrobenzylisoquinoline molecule on the way to norreticuline. One must postulate the existence of an enzyme which specifically methylates 6-0-methyl-norlaudanoso-line in the 4 -position with SAM serving as donor of the methyl group. Agmn,Berbers cell cultures (Hinz and Zenk 1981) proved to be an excellent plant source in the search for this new enzyme. Frenzel in our laboratory recently discovered this highly specific enzyme which catalyses the reaction depicted in Fig. 4. This enzyme should prove to be an interesting catalyst for specifically labelling norreticuline. 

The diagram shows that the catalytic action depends on a serine and a histidine residue. The histidine residue functions as a proton donor and acceptor. The reaction proceeds first by eliminating the group NH—X and by binding the acyl radical (the carboxyl group of the peptide bond under attack) to the enzyme, more specifically, to the serine residue. (Very careful hydrolysis has permitted the isolation of such serine-O-acyl compounds.) The ester bond is then hydrob zed the enzyme thus is returned to its original reactive form, and the second product of the hj drolysis, the acid, is liberated. The reactions are reversible to an extent depending on the equilibrium positions. 

The finding that CMP-sialic acid synthetase can accept many sialic acid analogs has facilitated the synthesis of CMP-sialic acid analogs for studies of the donor-specificity of these enzymes [88-90]. Both a2,3-SialT and a2,6-SialT tolerate substitutions at C-9 of CMP-sialic acid (Schemes 6 and 7) [89, 91, 92]. 

Blood groups A and B are synthesized by blood group A-dependent a3-GalNAc and blood group B-dependent a3-Gal-transferases, respectively. These two enzymes have similar acceptor substrate specificities and require the FI determinant as a substrate, while another a3-Gal-transferase, that is absent from humans and old world monkeys, acts on non-fucosylated terminal P-Gal residues and makes the linear B determinant, Galal-3Gaip- [89], The A and B transferases differ only by a few amino acids, which appear to determine the nucleotide-sugar donor specificities [90,91]. 

In 1987 Brozek et al., characterized an enzyme found in membranes of E. coli that catalyzes the transfer of a palmitate moiety from the sm-1 position of glycer-ophospholipids, such as phosphatidylethanolamine or phosphatidylglycerol, to the hydroxyl of the iV-linked acyl chain of lipid X [50], This enzyme is specific for glycerophospholipids as acyl donors. Acyl-ACP and acyl-CoA are not substrates [50]. The reaction is very specific for acyl chain length. No other fatty acyl chain can substitute for palmitate [50]. 

The first step in the catalytic cycle is nucleophilic addition of the anion of the cofactor thiamine diphosphate (ThDP) to the aldehyde. Deprotonation yields an enamine carbanion that serves as the nucleophile for the subsequent C—C bond-forming step. Alternatively, for the decarboxylase type ThDP-lyases, the carbonyl group of a 2-ketoacid donor substrate reacts with the anion of ThDP to yield the corresponding 2-hydroxy acid adduct. After CO is released, the enamine nucleophile is formed. Subsequently, the carbonyl acceptor reacts with the enamine to form the 2-hydroxy ketone. The stereoselectivity is governed by the enzyme that specifically discriminates between the two possible enantio-topic faces of the acceptor [14,48,49]. 

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