S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

Abstract

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His110 and His111) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His111 is unpaired, His110 is present as an ion pair with Asp22 on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P′3 position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His111 makes a positive 10-fold contribution to the exopeptidase activity, whereas His110 is critical for this action with the Asp22—His110 ion pair stabilizing the electrostatic interaction by a maximum of 13.9kJ/mol (3.3kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P′3 and its other subsites.

The Zoonotic Helminth Parasite Fasciola hepatica: Virulence-Associated Cathepsin B and Cathepsin L Cysteine Peptidases Secreted by Infective Newly Excysted Juveniles (NEJ)

“…The exopeptidase activity of the four recombinant peptidases was assayed against two fluorogenic exopeptidase substrates, Abz-Phe-Arg-Ala-Lys(Dnp)-OH (Exo-OH) and Abz-Phe-Arg-Ala-Lys(Dnp)-NH 2 (Exo-NH 2 ), designed to span the S2-S2′ subsites of the active peptidases [ 35 ]. Of the four F. hepatica cathepsin peptidases assessed, only the recombinant FhCB1 cleaved Exo-OH and Exo-NH 2 ; however, this activity was similar to that observed for SmCB1, but both were relatively low compared with the positive human control, HsCB ( Figure 4 ).…”

“…Cathepsin B peptidases are uniquely characterised by having a flexible loop structure, termed the occluding loop, which is located at the apical region of the active site cleft and confers these enzymes with unique carboxy-peptidyl-dipeptidase activity. This activity brings about the removal of two amino acid residues from the carboxy-terminus of protein substrates [ 24 , 35 , 46 , 47 ]. Deletion of the occluding loop sequence not only obliterates this exopeptidase activity but also increases the endopeptidase activity of the enzyme [ 48 ].…”

“…However, only His110 is thought to be critical for exopeptidase activity, while His111 increases the positive charge on the loop and increases the binding potential. To hold the occluding loop in an appropriate conformation for exopeptidase activity, in human cathepsin B, the His110 forms a salt-bridge interaction with Asp93, which is strengthened by the presence of His181 [ 35 ].…”

Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin

“…In human cathepsin B, two salt-bridge interactions between the occluding loop and the main body of the mature enzyme (Asp 22 -His 110 and Arg 116 -Asp 224 ) anchor the loop down in the active site, and unpaired His 111 forms a salt bridge with the C-terminus of an exo-substrate (Musil et al, 1991;Illy et al, 1997). According to another study (Krupa et al, 2002), His 111 is not critical for exopeptidase activity, although the level of this activity might be somewhat impaired by the absence of this residue. This means that also other substitutions in the sequence of TrCB1.6 are probably responsible for the disruption of exopeptidase activity in the reverse Gly 29 > Cys 29 mutant.…”

Organoruthenated Nitroxoline Derivatives Impair Tumor Cell Invasion through Inhibition of Cathepsin B Activity

“…The proteolytic activity of this enzyme is crucial in the mechanisms of cancer progression. Specifically, it has been identified as an important tumor-promoting factor − involved in extracellular matrix degradation, a process which enables tumor migration, invasion, metastasis, and angiogenesis. − CatB is unique in its structure among cysteine cathepsins by possessing an occluding loop, a 20 amino acid insertion, which defines whether the enzyme acts as an endopeptidase or an exopeptidase. − …”