Promoter site hydrolysis

Tryptophan 108 is recognized to be an active site in promoting the hydrolysis of 3(l,4)-glycosidic linkages between amino sugar residues in polysaccharide components of the bacterial cell walls. This residue is shown to occupy the cleft as well as trjrptophan 62 and 63, and is in a hydrophobic region. Tryptophan residues 62 and 108 are indispensable for the action of lysozyme, and tryptophan 62 is known to be the only binding site for the complex formation (13). Oxidation of tryptophan-108 is expected... 

Type I restriction enzymes, such as those encoded in the chromosome of E. coli, are large 300- to 400-kDa proteins composed of at least three kinds of polypeptide chain. They bind at specific sites of a foreign DNA and apparently cleave the chain randomly nearby. They require ATP, Mg2+, and S-adenosylmethion-ine and have the unusual property of promoting the hydrolysis of large amounts of ATP.81 82 The significance of these properties is still unknown. 

The prodrug methenamine, described above in this chapter (Scheme S-17), can be considered a site-specific chemical delivery system for the urinary tract antiseptic agent fotmal-dehyde. The low pH of the urine promotes the hydrolysis... 

The synthesis of [Zn(OH)(Tp Bu ,pr)], which has a structure very similar to the active site of the enzyme carbonic anhydrase, was described. [Zn(L)(Tp Bu, pr)] (HL = uracil or halouracil) were also reported. The halogenated uracil complexes were injected in Dalton s lymphoma tumor system in mice and [Zn(5-fluorouracilate)-(Tp Bu ,pr)] exhibits significant antitumor activity. This complex also promotes the hydrolysis of various esters and the maximum rate of hydrolysis is found with p-nitrophenylacetate.116... 

Following peptide bond synthesis, the ribosome Is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2-GTP. As a result of translocation, tRNAj , now without its activated methionine, is moved to the E (exit) site on the ribosome concurrently, the second tRNA, now covalently bound to a dIpeptIde (a peptIdyl-tRNA), Is moved to the P site (Figure 4-26, step U). Translocation thus returns the ribosome conformation to a state in which the A site Is open and able to accept another amlnoacylated tRNA complexed with EFlct-GTP, beginning another cycle of chain elongation. 

Inhibition Study. A proteinaceous inhibition study was conducted to study the role of the enzymatic active site in the hydrolysis and condensation of trimethylethoxysilane. Prior to reaction, trypsin was independently inhibited with an excess amount of the Bowman-Birk inhibitor (34) (4 1 BBI to trypsin mole ratio) and die Popcorn inhibitor (35) (2 1 PCI to trypsin mole ratio) in stirred neutral media for two hours. Based on standard enzymatic activity assays (36), trypsin was fully inhibited by the BBI (98%) and PCI (91%). The reactions were formulated with an 1000 1 trimethylethoxysilane to trypsin mole ratio and conducted at 25°C for three hours. The reaction products were isolated and quantitatively analyzed by GC (Table II). Although the treated enzymes were observed to catalyze the hydrolysis of trimethylethoxysilane, the condensation of trimethylsilanol was conqiletely inhibited in conq>arison to the control reactions. Notably, the rate of hydrolysis decreased in the presence of the BBI- and PCI-inhibited trypsin. Following thermal denaturation, tiie activity of trypsin was comparable to the proteinaceous inhibition experiments. Based on a standard enzymatic activity assay (36), the relative decrease in the rate of silanol condensation correlated with the enhanced stability of trypsin at higher protein concentrations (25). Consequently, it appears that non-specific interactions with trypsin including the active site promoted the hydrolysis of trimethylethoxysilane. Therefore, the active site of trypsin was determined to selectively catalyze the in vitro condensation of trimethylsilanol imder mild conditions. 

Followed by these observations, Bassindale et al. [ 19,20] studied the use of various homologous lipase and protease enzymes to catalyze the formation of molecules with a single siloxane bond during the in vitro hydrolysis and condensation of alkoxysilanes under mild reaction conditions. They found that non-specific interactions with trypsin promoted the hydrolysis of alkoxysilanes, while the active site was determined to selectively catalyze the condensation of silan-ols. One interesting observation was that when trypsin from various sources was employed different extents of conversion were observed. Comparatively, the activity of trypsin from a bovine pancreas was greater than the alternate sources of trypsin. Although various sources (e.g., mammalian, fish) of trypsin are similar (e.g., tertiary structure), their selectivity and activity was found to be different due to different optimum pH ranges and/or levels of calcium (an additive). 

Our parallel experiments, in which subtilisin Carlsberg was used to promote hydrolysis of A-acetyl-A-benzyl arenesulfinamides, led to exclusive S-N bond breaking. However, the recovered substrates were racemic. Moreover, blank experiments showed that a spontaneous chemical hydrolysis contributed to the process to a much higher degree than that in the cases shown in Ref. 47. Hence, a conclusion was drawn that in our case the hydrolysis proceeded without involvement of the subtilisin active site Kielbasihski, P. Albrycht, M. Mikolajczyk, M. Unpublished results. 

Inositol triphosphate (IP3)-gated channels are also associated with membrane-bound receptors for hormones and neurotransmitters. In this case, binding of a given substance to its receptor causes activation of another membrane-bound protein, phospholipase C. This enzyme promotes hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to IP3. The IP3 then diffuses to the sarcoplasmic reticulum and opens its calcium channels to release Ca++ ions from this intracellular storage site. 

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