Enzyme positional specificity

The classification adopted by the Nomenclature Committee (NC) of the International Union of Biochemistry and Molecular Biology (IUBMB) divides peptidases into classes and subclasses according to the positional specificity in the cleavage of the peptide link of the substrate. The last publication of the complete printed version of the Enzyme Nomenclature was in 1992 [1][2], but a constantly updated version with supplements is available on the World Wide Web at http //www.chem.qmul.ac.uk/iubmb/enzyme/. Similarly, all available Protein Data Bank (PDB) entries classified as recommended by the NC-IUBMB can be found on the WWW at http //www.bio-chem.ucl.ac.uk/bsm/enzymes/. 

The initial acylation at the 1-position of glycerol 3-phosphate is catalysed by glycerol 3-phosphate acyl-transferase-1, abbreviated to GPAT-1. This enzyme is specific for a saturated fatty acid (in the acyl form). 

It should be noted, however, that these enzymes catalyzed single methylation steps, and did not accept partially methylated substrates for further O-methylation. The common occurrence of partially methylated flavonoids (16,17), such as those of C. americanum (Figure 1), raised the question as to whether multiple methyl transfers were catalyzed by one or several position-specific O-methyltransferases ... 

Regulation of Flavonoid Synthesis in C. americanum. Biosynthesis of methylated flavonol glucosides seems to be under tight regulation, not only by the substrate specificity of the enzymes involved, but also by other factors, among which are (a) the strict position specificity of these enzymes towards their hydroxylated or partially methylated substrates (b) the apparent difference in microenvironment of the different methyl-transferases, whereby those earlier in the pathway utilized aglycones whereas later enzymes accepted only glucosides as substrates (c) the subtle characteristic differences in methyl-transferases with respect to their pH optima, pi values and requirement for Mg ions, despite their similar molecular size ... 

Double stranded DNA can be enzymatically cleaved by commercially available endonucleases (restriction enzymes). These enzymes recognise specific, usually palindromic, sequences and cut specifically at those positions. 

It has been proposed that 8-aminodcoxyguanosinc is formed from the nitronate tautomer of 2-nitropropane either by base nitrosation followed by reduction, or via an enzyme-mediated conversion of the nitronate anion to hydroxyiam ine-O-sulfonate or acetate, which yields the highly reactive nitrenium ion NHj (Sodum et al., 1993). Sodum et al. (1994) have provided evidence for the activation of 2-nitropropane to an aminating species by rat liver aryl sulfotransferase in vitro and in vivo. Pretreatment of rats with the aryl sulfotransferase inhibitors pentachlorophenol or 2,6-dichloro-4-nitrophenol significantly decreased the levels of nucleic acid modifications produced in the liver by 2-nitropropane treatment. Partially purified rat liver aryl sulfotransferase activated 2-nitropropane and its nitronate at neutral pH to a reactive species that aminated guanosine at the position. This activation was dependent on the presence of the enzyme, its specific cofactor adenosine 3 -phosphate 5 -phosphosulfate, and mercaptoethanol. It was inhibited... 

In agreement with the enzyme s specificity, acidic amino acids and N-alkylated amino acids afforded poor inhibitors. The discovery that lysine in the penultimate position provided good inhibition was at the time unprecedented in the ACE substrate or snake venom peptide literature, and this development was pursued with additional analogues (Table VI). Excellent activity was obtained with arginine and with higher and lower homologues of lysine. Even the s-N-acetyllysine analogue 33 (Table VI) was active. 

TOP) WHEN TWO PROTEINS BIND SPECIFICALLY, THEIR SHAPES MATCH EACH OTHER CLOSELY. (BOTTOM) TO CATALYZE A CHEMICAL REACTION AN ENZYME POSITIONS GROUPS CLOSE TO THE CHEMICAL THAT IT BINDS. THE SCISSORS REPRESENTS GROUPS ON THE PROTEIN THAT WILL CHEMICALLY CUT A SPECIFIC MOLECULE, REPRESENTED BY THE LIGHT-COLORED SHAPE. 

It has been reported that a microbial isolate, Flavobacterium sp. strain DS5, produced 10-ketostearic acid (10-KSA) from oleic acid in 85% yield (Hou, 1994a). The purified product was white, plate-like crystals melting at 79.2°C. A small amount of 10-hydroxystearic acid (10-HSA) was also produced during the bioconversion, suggesting that oleic acid is converted to 10-KSA via 10-HSA, and the enzyme catalyzing the hydration is C-10 positional specific (Hou, 1994b, 1995). The DS5 bioconversion products from oleic, linoleic, a-linolenic, and y-linolenic acid are all 10-hydroxy fatty acids. The optimum time, pH, and temperature for the production of 10-KSA have been reported in flask... 

Hou, C.T. 1995. Is strain DS5 hydratase a C-10 positional specific enzyme Identification of bioconversion products from a- and y-linolenic acids by Flavobacterium sp. DS5, /. Ind. Microbio., 14, 31-34. 

The positional specificity of Lipase B is questionable. The low activity of the immobilized enzyme, and consequently the low incorporation of lauric acid, makes the specificity determination uncertain. It is also difficult to determine the positional specificity of Lipase B before irrmobilization by the traditional analysis of 1,2- and 1,3-diolein obtained by hydrolysis of triolein as the diolein is very quickly degraded by the enzyme. 

The esterases are involved in the hydrolysis of ester linkages of various types. The products formed are acid and alcohol. These enzymes may hydrolyze triglycerides and include several lipases for instance, phospholipids are hydrolyzed by phospholipases, and cholesterol esters are hydrolyzed by cholesterol esterase. The carboxylesterases are enzymes that hydrolyze triglycerides such as tributyrin. They can be distinguished from lipases because they hydrolyze soluble substrates, whereas lipases only act at the water-lipid interfaces of emulsions. Therefore, any condition that results in increased surface area of the water-lipid interface will increase the activity of the enzyme. This is the reason that lipase activity is much greater in homogenized (not pasteurized) milk than in the non-homogenized product. Most of the lipolytic enzymes are specific for either the acid or the alcohol moiety of the substrate, and, in the case of esters of polyhydric alcohols, there may also be a positional specificity. 

A combination of the specific enzymes with the sensitivity, speed and accuracy of mass spectrometry allows complete sequence determination, including the anomeric configuration. According to the specificity of the enzymes, positional isomers of the glycosidic bonds can be recognized. 

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