Hydrolysis sites

During polyadenylation the primary transcript is shortened in an endonucleolytic step and appended with ca. 200 A-residues. The endonucleolytic incision requires two signal sequences on the pre-mRNA. A highly conserved AAUAAA sequence 10-30 nucleotides upstream from the hydrolysis site serves as one signal. Another signal in the form of a less well conserved GU- or U-rich element upstream of the hydrolysis site. Both together constitute the polyadenylation signal (Fig. 1.45). Polyadenylation occurs in a multiprotein complex, whose composition is not yet explained in all details. 

Phospholipase C (PL-C) is activated by G-protein a- and/or j3y-subunits. The hydrolysis site is indicated by the open arrow. Two products are obtained 1,2-diacylglycerol (DAG) which activates protein kinase C and inositol 1,4,5-trisphosphate (IP3) which induces Ca2+ release from intracellular stores. 

Figure 1. Some restriction endonucleases hydrolysis sites on...

None of the exopeptidases or endopeptidases that we have mentioned will catalyze the hydrolysis of an amide bond if proline is at the hydrolysis site. These enzymes recognize the appropriate hydrolysis site by its shape and charge, and proline s structure causes the hydrolysis site to have an unrecognizable three-dimensional shape. 

Serine proteases do not catalyze hydrolysis if the amino acid at the hydrolysis site is a D-amino acid. Trypsin, for example, cleaves on the C-side of L-Arg and L-Lys, but not on the C-side of D-Arg and D-Lys. Explain. 

Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y. Y., and Sutoh, K. (2004) Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. Biochemistry 43, 11266-11274. 

The following is a section of the gene coding for bovine rhodopsin, along with a table listing several restriction endonucleases, their recognition sequences, and their hydrolysis sites ... 

Comparison of the structures of bovine trypsinogen and chymotrypsinogen A. Solid arrows show the hydrolysis sites that result in the activation of trypsinogen to trypsin, and chymotrypsinogen to chymotrypsin. Broken arrows are additional hydrolysis sites, leading to formation of a-chymotrypsin. Shaded circles represent amino acids that are Identical or similar in the two proteins. Disulfide bridges are lettered A to G. H represents the histidine residues, and S the serine residue of the active site this serine residue reacts with diisopropylfluorophosphate. Deletions are shown by lines between the circles. To aid comparison, the residue numbers of both structures are based on those of chymotrypsinogen. [B.S.Hartley etal. Nature 207 (1965) 1157-1159]... 

Nontoxic degradation by-product (lysine) Methyl ester provides steric hindrance of hydrolysis sites [60,125,140,141,143,160]... 

Fig. 5. Substrate specificities of CBH I, CBH II, EG I and EG III from T. reesei, using low molecular weight derivatives of the cellooligosaccharides. Symbols czi, 1,4-D-glucopyranosyl A, 1,4-D-galactopyranosyl El, reducing end 0,, 4-methyl-umbelliferyl- the arrow indicates the hydrolysis site, and the number specifies the turn over number (min ) not determined. Taken from Refs, [101, 149, 200], by permission...

Researchers at different companies used different reasoning in developing drugs. For example, chemists at Hoffinan-La Roche imitated the hydrolysis site of the gag-pol protein, that is, Phe-Pro (see above). They modified the surroundings of the site and discovered that saquinavir is the most effective (see the structure in Fig. 7.4). In Abbot s Lab, the scientists made use of the symmetrical nature of the HlV-1 protease. Because most of other mammalian proteases are not symmetrical, a compound with a symmetrical structure about the mimicking site would inhibit the HlV-1 protease, but not other mammalian proteases, thus hopefully reducing side effects. With this notion in mind, they synthesized a number of compounds and discovered that ritonavir was most effective (see the structure in Fig. 7.4). As you see in Fig. 7.4, the... 

Cyanogen bromide (BrC=N) hydrolyzes the peptide bond on the C-side of methionine. Cyanogen bromide is more specific than the endopeptidases about which peptide bonds it cleaves, so it provides more reliable information about the primary structure. Because cyanogen bromide is not a protein, it does not recognize the substrate by its shape. As a result, it will still cleave the peptide bond if proline is at the hydrolysis site. 

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