Hydrolytic sites

Fig. 3.17. The crystal structure of human serum albumin (HSA) complexed with four molecules ofmyristic acid (from lbj5.pdb [121][122]). The picture shows the domains (I—III) and subdomains (A and B) of HSA. The primary hydrolytic site is located in subdomain IIIA, and two others probably in subdomain IIA. 

Figure 13.2 A possible specificity mechanism for the prevention of the misacylation of tRNAVal with threonine, (a) The hydrophobic acylation site discriminates against threonine, (b) The hydrolytic site specifically uses the binding energy of the hydroxyl of threonine for a binding or catalytic effect. The translocation may occur as illustrated via a 2 - — 3 -hydroxyl acyl transfer. [From A. R. Fersht and M. Kaethner, Biochemistry 15, 3342(1976).]...

Figure 13.4 The double sieve analogy for the editing mechanism of the isoleucyl-tRNA synthetase. The active site for the formation of the aminoacyl adenylate can exclude amino acids that are larger than isoleucine but not those that are smaller. On the other hand, a hydrolytic site that is just large enough to bind valine can exclude isoleucine while accepting valine and all the smaller amino acids. (In some enzymes, the hydrolytic site offers specific chemical interactions that enable it to bind isosteres of the correct amino acid as well as smaller amino acids.)...

Uracil is removed from the DNA by a uracil glycosidase which excises the base from the sugar ring. This activity is analogous to the hydrolytic activity of the isoleucyl-tRNA synthetase toward Val-tRNAIle. In both cases the hydrolytic site is too small by the size of one methylene group to accommodate the substrate that is to be left intact. In DNA synthesis, the editing is performed by a separate enzyme, since the editing can wait until after polymerization. As this luxury is not permitted in protein synthesis, the hydrolytic function is on the synthetase, so that correction can occur before the misacylated tRNA leaves the enzyme. 

J. J. Hopfield has suggested a general mechanism called kinetic proofreading in which there is no hydrolytic site on the enzyme instead, the desired intermediates diffuse into solution, where they hydrolyze nonenzymatically.54 An example is in the selection of amino acids by the aminoacyl-tRNA synthetases (equation 13.32). 

Suggestions concerning the structural element to which the enzyme binds vary. Razzell and Khorana (51), on the basis of minimal requirements for activity, considered the binding site identical with the hydrolytic site, which is limited to one doubly esterified 5 -nucleotide. Bjork (43) believed that besides a hydrolytic site additional binding sites exist. In this connection it should be mentioned that whereas a 5 -mono-... 

This finding indirectly supports Bjork s hypothesis regarding the existence of multiple sites of attachment of substrate to enzyme. The absence of the 5 -monophosphoryl group is felt by the enzyme even though the hydrolytic site is removed from the a terminus by several monomers. 

One particularly well-understood ATP-dependent protease is the La protease that is the product of the Ion gene of E. coli. This protease, like many ATP-dependent proteases, is a large protein, and its ability to hydrolyze proteins is tightly coupled to its ability to hydrolyze ATP. Approximately two ATPs are hydrolyzed to ADP and P, for each peptide bond that is hydrolyzed. It appears that the hydrolysis of ATP is required to activate the proteolytic active site of La. Other proteases seem to require ATP as an allosteric effecter to activate their hydrolytic sites, but this ATP is not hydrolyzed. 

Figure 5.13 Interaction of an amylose chain in the vicinity and in the hydrolytic site of pig pancreatic a-amylase. The impossibility of fitting a double helix in the hydrolytic site has been clearly established, along with determination ofthe direction of binding of the amylosic substrate in the cleft of the enzyme. (Reproduced with permission from reference 51)...

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

Most aminoacyl-tRNA synthetases contain editing sites in addition to acylation sites. These complementary pairs of sites fimction as a double sieve to ensure very high fidelity. In general, the acylation site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller than the correct one. 

Lipases are ester hydrolases acting on triacylglycerols. They achieve their highest catalytic rate at oil-water interfaces. Though they widely differ in size, substrate and catalytic rate and show little sequence similarity, the Ser...His...Asp(Glu) triad in the active site is a structural feature common to serine proteases (cf. Section 3.1.1.) and lipases. Their mechanism of action is not yet fully revealed. X-ray diffraction studies suggest that the putative hydrolytic site is covered by a surface loop and is therefore inaccessible to solvent (Brady et al., 1990 Winkler et al., 1990). Therefore the enzyme... 

The toxicity of the G and V agents is apparently due to their affect upon the transmission of nerve impulses in the body, due to their ability to react with and inhibit acetylcholinesterase (ChE) enzyme. For example, after a nerve impulse is transmitted across a nerve and muscle junction by acetylcholine, the latter is then rapidly hydrolyzed by ChE. However, in the presence of GB, for example, the hydrolytic sites on the enzyme are irreversibly blocked by phosphorylation, and these sites are no longer available for hydrolysis of the acetylcholine. The resulting buildup of acetylcholine acts to overstimulate the muscle, which eventually goes into spasm. Thus, the typical symptoms of poisoning by a nerve agent are due to its cholinergic activity. 

The immobilized F-II cleaves human plasminogen at four hydrolytic sites Lys77-Arg7g, Arg342-Met343, Alam-Ala s and Arg557-Ile558... 

The ATP hydrolytic sites for the proton pump are located at cytosolic sites and the high affinity K+ sites are on luminal face across the membrane [140]. The enzyme is phosphorylated at cytosolic sites by ATP in the presence of Mg2+. Then the enzyme-phosphate complex is dephosphorylated by luminal K+. The kinetic studies carried out by Murakami et al. [139,141] demonstrated that the inhibition of the gastric pump by sofalcone, chalcone and quercetin was competitive with respect to ATP and noncompetitive with K+. In this way, Beil et al. [142] showed that quercetin, flavone and flavanone locked acid formation in parietal cells in response to histamine and cAMP stimulation, flavanone being the most potent inhibitor. H+, K+-ATPase was inhibited by all of them, and this inhibition increased with lowering ATP concentration. The steady-state phosphorylation level of the enzyme was also dose-... 

Proofreading in amino acid activation takes place in two stages. The first requires a hydrolytic site on the aminoacyl-tRNA synthetase incorrect amino acids that have become esterified to the tRNA are removed here. The second stage of proofreading requires the recognition site on the aminoacyl-tRNA synthetase for the tRNA itself. The incorrect tRNA does not bind tightly to the enzyme. 

Threonyl-tRNA synthetase has a proofreading mechanism. Any Ser-tRNA that is mistakenly formed is hydrolyzed by an editing site 20 A from the activation site. The decision to hydrofyze the aminoacyl-tRNA appears to depend on the size of the amino acid substituent, ff it is smaffer than the correct amino acid, the amino acid fits into the hydrolytic site and is cleaved. If it is the same size as the correct amino acid, it does not fit and is not destroyed. Discrimination between amino acids that are larger than the correct one or are not isoelectronic with it occurs at the aminoacylation step. 

Oligo-1,6-D-glucosidase-sucrose a-D-glucohydrolase ( sucrase-isomaltase ) is an intestinal membrane enzyme consisting of two active moieties, each with its hydrolytic site available for nutrient digestion at the luminal-cell interface. 

To answer these questions, we have analyzed the frequency of appearance of various amino acid residues at the loci near the sessile bond in protein substrates (4). This was statistically analyzed for more than 500 amino acid sequences in which the peptic hydrolytic sites are known. The calculated specificity indices, Sij for some of the residues (i) and positions (j) are shown in Table I. This index is a measure of the preferability, undesirability, or indifference for a given amino acid side chain. The calculation included ten residues on each side of the hydrolyzed bond. We also... 

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