Enzyme substrates, synthesis

Product Enzyme Substrate Synthesis Strategy Reference... 

In biological systems molecular assemblies connected by non-covalent interactions are as common as biopolymers. Examples arc protein and DNA helices, enzyme-substrate and multienzyme complexes, bilayer lipid membranes (BLMs), and aggregates of biopolymers forming various aqueous gels, e.g, the eye lens. About 50% of the organic substances in humans are accounted for by the membrane structures of cells, which constitute the medium for the vast majority of biochemical reactions. Evidently organic synthesis should also develop tools to mimic the Structure and propertiesof biopolymer, biomembrane, and gel structures in aqueous media. 

A pepsin hydrolysate of flounder fish protein isolate has been used as the substrate (40% w/v) for plastein synthesis, using either pepsin at pH 5 or alpha chymotrypsin at pH 7, with an enzyme—substrate ratio of 1 100 w/v at 37°C for 24 h (151). The plastein yields for pepsin and alpha chymotrypsin after precipitation with ethanol were 46 and 40.5%, respectively. 

Boyd DR, ND Sharma, MV Hand, MR Groocock, NA Kerley, H Dalton, J Chima, GN Sheldrake (1993) Stereodirecting substituent effects during enzyme-catalysed synthesis of cw-dihydrodiol metabolites of 1,4-disubstituted benzene substrates. J Chem Soc Chem Commun 974-976. 

PK Banerjee, GL Amidon. Physicochemical property modification strategies based on enzyme substrate specificities I Rationale, synthesis, and pharmaceutical properties of aspirin derivatives. J Pharm Sci 70 1299, 1981. 

Guzikowski AP, Naleway JJ, Shipp CT, Schutte RC (2000) Synthesis of a macrocyclic rhodamine 110 enzyme substrate as an intracellular probe for caspase 3 activity. Tetrahedron Lett 41 4733 1735... 

Figure 9.9 Kinetic scheme for MBI.

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

Fig. 7.21. Activation of glycogen-bound protein phosphatase I by insulin. Insulin has a stimulating effect on glycogen synthesis by initiating the dephosphorylation and activation of glycogen synthase and the dephosphorylation and inhibition of glycogen phosphorylase. Both enzymes (substrate S in the figure) are dephosphorylated by protein phosphatase PPIG. Insulin mediates the activation of a protein kinase (insulin-sensitive protein kinase) within an insulin-stimulated signal pathway, which phosphorylates and thus activates protein phosphatase PPIG at the PI site.

Enzyme inhibition. The enzymes of biotransformation may be inhibited by a single exposure to chemicals. This occurs by several mechanisms formation of a complex, competition between substrates, destruction of the enzyme, reduced synthesis of the enzyme, allosteric effects, and lack of cofactors. The consequences will depend on the role of metabolism in toxicity in the same way as induction (see above). 

The mode of action of the sulfonamides as antagonists of 4-aminobenzoic acid (PAB) is well documented, as is the effect of physicochemical properties of the sulfonamide molecule, e.g. pK, on potency (B-81MI10802). Sulfonamides compete with PAB in the biosynthesis of folic acid (44), a vital precursor for several coenzymes found in all living cells. Mammalian cells cannot synthesize folic acid (44), and rely on its uptake as an essential vitamin. However, bacteria depend on its synthesis from pteridine precursors, hence the selective toxicity of sulfonamides for bacterial cells. Sulfonamides may compete with PAB at an enzyme site during the assembly of folic acid (44) or they may deplete the pteridine supply of the cell by forming covalently-bonded species such as (45) or they may replace PAB as an enzyme substrate to generate coupled products such as (46) which are useless to the cell. 

Extensive studies of enzyme-substrate complexes by resonance Raman spectroscopy (RR) have prompted the synthesis of new peptide bond modifications such as thionoesters and dithioesters (Scheme l7)t82-83l within simple model substrates. The resulting acyl-enzyme complexes are especially amenable to RR analysis with cysteine proteases such as papain due to formation of the transient dithioester intermediates. 

In more recent investigations, the assumed multienzyme involved in cyclosporin A biosynthesis could be isolated from T. inflatum. A partially purified enzyme fraction was indeed capable of forming enzyme-substrate complexes by thioester linkage. Although de novo synthesis (in vitro) of cyclosporin A has not yet been achieved, the formation of a partial sequence, namely, the diketopiperazine cyclo(DAla-MeLeu), from D-alanine and L-leucine was observed under consumption of ATP and S-adenosyl-L-methio-nine [25]. 

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