Aspartate substrate specificity

Graf, L., et al. Selective alteration of substrate specificity by replacement of aspartic acid 189 with lysine in the binding pocket of trypsin. Biochemistry 26 ... 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Aminopeptidase A is another brush border membrane enzyme which has been the subject of various studies [79,81,83-86], It has been found in the intestinal brush border membrane of humans, rabbits, rats, and pigs and is active against peptides with acidic amino acids at the amino terminus. Its activity against dipeptides is more limited. Shoaf et al., isolated three rat brush border aminopeptidases with distinct but somewhat overlapping substrate specificities. These enzymes had preference for dipeptides containing methionine, arginine, or aspartic acid and glycine. The optimal pH for activity of aminopeptidase was reported to be 7-8. 

Paine MJ, McLaughlin LA, Flanagan JU, et al. Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6. J Biol Chem 2003 278(6) 4021-4027. 

In this transamination, the effect of para substitient groups has been studied using fluorinated phenylpyruvic acids and L-aspartic acid. From these results, the migratory preference is H > F > Cl > Br > CF3. This order has been attributed to the bulkiness of the substituted group [57]. Direct amination of p-substituted succinic acid with phenylalanine ammonialyase (EC 4.3.1.5) has suggested very high substrate specificity that the order of reaction rate is m-F o-F P-p-F >CF3. 

Substrate specificity of aminotransferases Each amnolrans ferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino gap donor, because the acceptor of the amino group is almost always a-ketoglutarate. The two most important aminotrans ferase reactions are catalyzed by alanine aminotransferase ati aspartate aminotransferase (Figure 19.8). 

Many enzymes exist within a cell as two or more isoenzymes, enzymes that catalyze the same chemical reaction and have similar substrate specificities. They are not isomers but are distinctly different proteins which are usually encoded by different genes.22 23 An example is provided by aspartate aminotransferase (Fig. 2-6) which occurs in eukaryotes as a pair of cytosolic and mitochondrial isoenzymes with different amino acid sequences and different isoelectric points. Although these isoenzymes share less than 50% sequence identity, their internal structures are nearly identical.24-27 The two isoenzymes, which also share structural homology with that of E. coli,28 may have evolved separately in the cytosol and mitochondria, respectively, from an ancient common precursor. Tire differences between them are concentrated on the external surface and may be important to as yet unknown interactions with other protein molecules. 

If a lysine were substituted for the aspartate in the trypsin side-chain-binding crevice, would you expect the enzyme to be functional If it were functional, what effect would you predict the substitution to have on substrate specificity ... 

Brinkworth, R.I., Prociv, P., Loukas, A. and Brindley, P.J. (2001) Haemoglobin-degrading, aspartic protease of blood-feeding parasites substrate specificity revealed by homology models. The Journal of Biological Chemistry 276, 38844-38851. 

J. J. Onuffer and J. F. Kirsch, Redesign of the substrate specificity of Escherichia cdi aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis, Protein Sci. 1995, 4, 1750-1757. 

E. Sandmeier, E. Marra, and P. Christen, Active-site Arg->Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase, J. Biol. Chem. 

T. Yano, S. Oue, and H. Kagamiyama, Directed evolution of an aspartate aminotransferase with new substrate specificities, Proc. Natl. Acad. Sci. USA... 

In order to obtain further information about the primary specificity of aspergillopepsin I, we constructed an aspergillopepsin I expression system in Escherichia coli, prepared mutants at positions 76 and 78 of aspergillopepsin I by site-directed mutagenesis, and compared then-molecular and enzymatic properties [30], We showed that the nature of the active site flap is important for deciding the Si substrate specificity of aspartic proteinases [30],... 

Pepsin (EC 3.4.23.1) is a typical aspartic proteinase produced in the gastric mucosa of vertebrates as a zymogen form [10], This enzyme has been extensively characterized, and its three-dimensional structure has been determined at high resolution. Porcine pepsin, in particular, has been studied as model to analyze the structure-function relationship of the aspartic proteinases. Although the aspartic proteinases including mammalian and fungal enzymes are quite similar in their three-dimentional structures, there are drastic differences in the catalytic properties, especially in substrate specificities. 

Alteration of Si Substrate Specificity of Pepsin to those of Fungal Aspartic Proteinase... 

In conclusion, the double mutant pepsin T77D/G78(S)S79 was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contribute to the Si substrate specificity for basic amino acid residues in aspartic proteinases. 

Shintani, T., Nomura, K., and Ichishima, E. (1997). Engineering of porcine pepsin Alteration of Si substrate specificity of pepsin to those of fungal aspartic proteinase by site-directed mutagenesis. J. Biol. Chem., 272, 18855-18861. 

Aspartase exhibits incredibly strict substrate specificity and thus is of little use in the preparation of L-aspartic acid analogues. However, a number of L-phenylalanine analogues have been prepared with various PAL enzymes from the yeast strains Rhodotorula graminis, Rhodotorula rubra, Rhodoturula glutinis, and several other sources that have been cloned into E. call.243 241 Future work in this area will likely include protein engineering to design new enzymes that offer a broader substrate specificity such that additional L-phenylalanine analogues could be prepared. 

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