Proteases amino acid sequence

Table 7-1. Amino acid sequences in the neighborhood of the catalytic sites of several bovine proteases. Regions shown are those on either side of the catalytic site seryl (S) and histidyl (H) residues.

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

Fig. 6. Terminal capping and lateral bulging of globular domains in the //-solenoid of the hemoglobin protease from E. coli (Otto et al., 2005). The //-solenoid domains are shown in blue and the remaining regions in dark yellow. (A) Ribbon diagram of the 3D structure and (B) linear map of the domain distribution within the amino acid sequence.

Fig. 8. Organization of the kringle IV repeats in apo(a). It has been determined that kringle IV repeats, which vary in amino acid sequence from the identically repeated kringle sequence (kringle IV types 1 and 3-10), are highly variable in the population, ranging from less than 10 to greater than 50. The numbers in brackets below the boxes refer to the kringle nomenclature described in McLean et at. (M24). Sequences corresponding to apo(a) kringle V and protease domains are indicated by stippled and solid boxes, respectively.

Thermolysin is a single-chain protease composed of 316 amino acids its amino acid sequence and three-dimensional structure (Colman et at, 1972) have been determined. The native protein has no disulfide bonds, but does contain both Ca2+ and Zn2+, which strongly stabilize the structure. 

Figure 3. Structure of IgAj and the amino acid sequence of the hinge region showing sites of cleavage by various IgAj proteases.

Several different proteases can attack a single protein at enzyme-selective amino-acid sequences. Proteases can be divided into two categories. Endopeptidases are enzymes that cleave peptide bonds between specific, nonterminal amino acids. There are endopeptidases specific for just about every amino acid. Exopeptidases are enzymes that cleave terminal peptide bonds at either the C-terminus or N-terminus. 

Shark meat hydrolyzed with protease SM98011 showed high ACE inhibitory activity, with an IC50 value of 0.4mg/ml (He et ah, 2007). Four peptides with high ACE inhibitory activity were purified from shark meat hydrolysate. Their amino acid sequences were CF, EY, MF, and FE, and their IC50 values were 1.98, 2.68, 0.92, and 1.45mM, respectively. They may have potential in the treatment of hypertension or in clinical nutrition (Wu et ah, 2008). 

Among proteases, the digestive enzyme trypsin catalyzes the hydrolysis of only those peptide bonds in which the carbonyl group is contributed by either a Lys or an Arg residue, regardless of the length or amino acid sequence of the chain. The number of smaller peptides produced by trypsin cleavage can thus be predicted... 

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