Acid proteases sequence studies

G, Kamphuis, J. Drentii, and E. N. Baker. Thiol protease. Comparative studies based on the high-resolution structures of papain and actinidin, and an amino acid sequence information far cathenaim B and R and stem bromelain. J. MoL BioL /62 317 (19B5). 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

An archaebacterial acid protease (optimum pH 2.0) which may represent a new class of enzyme has been purified recently from Sulfolobus acidocaldarius [297, 298]. Although the specificity of the enzyme was similar to pepsin, with preference shown towards bonds between large hydrophobic residues, inhibitor studies indicated differences in active site composition. Furthermore, the nucleotide sequence of the enzyme gene was completely dissimilar to those for enzymes of the pepsin family and to pepstatin-insensitive acid protease. 

To localize the rate of deuterium buildup to specific amides, the analyte protein is fragmented into a collection of peptides using combinations of endo- and exoproteases. Due to the low pH of the quench conditions in which the protein and peptide samples are maintained after deuterium labeling, acid-reactive proteases such as pepsin must be employed. Studies with combinations of acid-reactive en-doproteinases and carboxypeptidases have been employed to achieve greater sequence coverage and higher amide resolution [42, 45]. 

From study of peptides formed by partial hydrolysis of the 32P-labeled chymotrypsin, the sequence of amino acids surrounding the reactive serine was established and serine 195 was identified as the residue whose side chain hydroxyl group became phosphorylated. The same sequence Gly-Asp-Ser-Gly was soon discovered around reactive serine residues in trypsin, thrombin, elastase, and in the trypsin-like cocoonase used by silkmoths to escape from their cocoons.198 We know now that these are only a few of the enzymes in a very large family of serine proteases, most of which have related active site sequences.199 200 Among these are thrombin and other enzymes of the blood-clotting cascade (Fig. 12-17), proteases of lysosomes, and secreted proteases. 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

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