Proline residues substrate specificity

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

These features of substrate specificity of tTG are mirrored by epitope specificity of gliadin antibodies of CD patients. The common core of the nona-peptides specifically recognized by antibodies of CD patients is QPEQPFP with the amino acid proline residing in position +2 [127]. However, in accordance with results from studies of substrate specificity of tTG [182], exchange of phenylalanine residue for tyrosine completely abolished binding of IgA of CD patients [127]. 

Elastase is the name given to proteinases that possess the ability to hydrolyze mature cross-linked elastin [18]. Elastin is an insoluble structural protein responsible for the elastic properties of the lung, skin, and arteries and is quite resistant to most proteinases. Elastin is high in hydrophobic amino acid residues such as valine, alanine, glycine, and proline [19]. Insoluble elastin fibers contain cross-links usually between four lysine residues, which form a unique cyclic product, desmosine. The presence of soluble desmosine cross-links in plasma can be used as a measure of elastin breakdown. Of all the elastases in humans, neutrophil elastase has received the most attention over the years due to its broad substrate specificity and abundance within the cell. However, neutrophils and macrophages contain several proteinases (Table 1), which are capable of degrading elastin. 

There are some interesting differences between the liver and yeast enzymes among the residues which are in the active site pocket region. Residues 58 and 93 are both substituted by tryptophans in the yeast enzyme which narrows the pocket. Furthermore, the two prolines which are inserted on both sides of residue 58 in the yeast enzynie could diminish the pocket even further. A smaller active site pocket in the yeast enzyme is consistent with the substrate specificity of the two enzymes (Section III,E) as well as the ability to bind big chelating molecules like 1,10-phenanthroline (Section IH,D). 

Prolyl hydroxylase from the earthworm requires the same co-factors as the animal enzymes, but it has a different substrate specificity. It requires that the glycine residue precede the proline rather than follow it. Thus, the peptide (Gly-Pro-Ala)n is a substrate for earthworm prolyl hydroxylase but not for mammalian and bird enzymes (132). Prolyl hydroxylase from carrots appears to be very similar in co-factor requirements and specificity to that of the animal enzymes (119). The plant enzyme could hydroxylate collagen prepared from chick-embryo tibias and the animal enzyme could hydroxylate the unhydroxylated plant substrate dehydroxyextensin. 

Staphylococcus aureus V8 protease deaves proteins specifically after glutamate residues, provided that the following residue is not proline, or a further glutamate or aspartate. In ammonium bicarbonate buffer at pH 7.8, the enzyme is very specific for glutamate in Na-phosphate buffer at the same pH it also cleaves adjacent to aspartate. V8 protease is added at a ratio of 1-2% (w/w) to the protein substrate. Reaction can be stopped by freezing, or by the addition of PMSF or a2-rtiacroglobu-lin. 

Aminopeptidases are present in many tissues (Table III). Leucine aminopeptidase from intestinal mucosa is very effective in catalyzing the hydrolysis of leucine from the amino terminus of peptides, polypeptides, and proteins. It also hydrolyzes leucine amide and leucine esters (10). The designation leucine aminopeptidase is somewhat of a misnomer because activity is also observed when other amino acids replace leucine. Only the L-isomers of amino acids are substrates, and the presence of a D-amino acid residue or proline in the penultimate position will retard hydrolysis (10). Enzymes having the same specificity as the intestinal aminopeptidase have been identified and/or isolated from kidney, pancreas, muscle, lens, and various bacterial sources (10). The kidney... 

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