Substrate profile

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

Figure 2.12 Substrate profiles of lipase variants produced by CASTing [25].

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

Figure 5.253. A plot of the substrate profiles indicate substantial gradients, but the substrate concentrations are not low enough to influence the rates.

Yang, Y., Zhu, D., Piegat, T.J. and Hua, L. (2007) Enzymatic ketone reduction mapping the substrate profile of a short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae. Tetrahedron Asymmetry, 18 (15), 1799-1803. 

FIGURE 2.8. Substrate profiles exemplified for a biofilm with full and part penetration of a single substrate. 

The carbon-carbon forming ability of aldolases has been limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has most often not produced the desired effect. Directed evolution approaches have furnished novel activities through multiple mutations of residues involved in recognition in no instance has a key catalytic residue been altered while activity is retained. Random mutagenesis resulted in a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile (Wymer, 2001). 

As increasing research has been carried out with these enzymes, a less empirical approach has been taken as a result of the different substrate profiles that have been compiled for various enzymes in this class. These profiles have been used to construct active site models for such versatile enzymes as the carboxylester hydrolase, pig liver esterase (PLE) (E.C. 3.1.1.1), and the microbial lipases (E.C. 3.1.1.3) from Burkholderia cepacia (formerly Pseudomonas cepacia) lipase (PCL), Candida... 

Fed-batch cultivation (A) operational configuration and typical volume, cell, and substrate profiles, for a pulse feeding strategy (B) concentration of cells, product, and two substrates in a fed-batch bioreactor culture of a myeloid transfectoma producing a humanized monoclonal antibody (Center of Molecular Immunology-CIM, Cuba). 

When attention is directed toward the phospholipase.C found in mammalian tissue, a rather unique and different substrate profile is evident. It appears that the most favored substrate status must be assigned to the inositol- containing phosphoglycerides, namely, phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). There is some evidence that the plasma membrane of certain mammalian cells contains a phospholipase C with high specificity for the bisphosphate, PIP2. The latter enzymatic interaction would be closely associated with the signal transduction pathway in mammalian cells. 

The product z (r) reaches the maximum plateau level asymptotically. In contrast to the substrate profile, the nonlinear behavior along the saturation of the complex is not easily defined on the product profile. 

Figure 8.4 shows the influence of e on the x (r) shape. For fixed (k, A), we simulated the time courses for e = 0.5, 1, 2, 5. It is noted that the shape of the substrate profiles varies remarkably with the values of e thus profiles of biphasic, power-law, and nonlinear type are observed. So, the sensitivity of the kinetic profile regarding the available substrate and enzyme amounts is studied by using several e values for low substrate or high enzyme amounts the process behaves according to two decaying convex phases, in the reverse situation the kinetic profile is concave, revealing nonlinear behavior. 

MENG, H., CAMPBELL, W.H., Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development, Plant Mol. Biol., 1998, 38, 513-520. 

Lactamases are heterologous in nature both in terms of their chemical structure and their substrate profile [181] (Table 4.13). The original Rich-mond-Sykes [182] classification of -lactamases produced by Gram-negative bacteria envisaged five classes of enzymes. Of these, class I had a cephalo-... 

TABLE 69.2. Substrate profile of enzymes with potential use as protective bioscavengers... 

Figure 24.4 Schematic diagram of potential N substrate profiles in a stratified water column, mapped against the known partitioned distribution of specific Prochlorococcus genotypes/ecotypes from molecular ecological studies (e.g., see Moore et ah, 1998 Steglich et ah, 2003 West and Scanlan 1999).

Pratt MR, Hang HC, Ten Hagen KG, Rarick J, Gerken TA, Tabak LA, Bertozzi CR. Deconvoluting the functions of polypeptide W-alpha-acetylgalactosaminyltransferase family members by gly-copeptide substrate profiling. Chem. Biol. 2004 11 1009-1016. 

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