It is resistant to most protease inhibitors, given the steric restrictions provided by the narrow active sites all facing the central pore. As such, the question of regulation arises, since the fully formed protein cannot be inhibited endogenously in Homo sapien. In contrast to the alpha tryptase protein, which shares 90% similarity with beta tryptase, the latter cannot exist as a tetramer in the absence of heparin, making the only viable biologic regulation pathway at this stage of protein maturity the slow restriction of heparin. Since the beta tryptase monomer displays no enzymatic or catalytic activity, the pH-dependent dissociation of heparin is the predominant protein regulation mechanism in vivo.
Beta-tryptase is a tetramer with two sets of homodimeric subunits, arbitrarily labeled as A, B, C, and D by scientists. A/C and B/D are identical, and the only difference between the two pairs is the orientation of the Tyr-75 side chain. The tetramer weights 110.41 kDa with 7,872 total atoms across the four monomers, each 244 amino acids in length with alternating polar and nonpolar regions, with gray regions denotating an unspecified charge. The monomers are not covalently bound to each-other and are instead stabilized by heparin (a large and highly electronegative polysaccharide more commonly known for its role as an anticoagulant). The active sites of each subunit face inwards, limiting the possible substrates and reactions. Each monomer is comprised of two alpha helices and beta barrels, with the aforementioned active site occuring directly between the two barrels. Beta-tryptase preferentially severs polypeptide chains at arginine and lysine residues as a serine protease with an active site at amino acid histodine 57, aspartate 102, and serine 195.
The aforementioned monomers interact with each-other via polarity-dependent mechanisms for six different loops in the active site — loops 37, 60, 70-80, 97, 147, and 173. Although beta tryptase shares many sequence similarities to trypsin or chymotrypsinogen, the main differences originate from added amino acids in the loops, enabling the former protein to form a tetramer and bind substrates such as vasoactive intestinal peptide (VIP) or proteinase-activated receptor (PAR2). Homodimers A/B or C/D interact through loops 147, 70-80, and 37. Additionally, the hydrophobic 152 spur fits into a gap formed by loops 37 and 70-80 of its monomer and the spur of its neighboring subunit. This nonpolar interaction is induced by the high concentration of proline residues in the spur, which is also visible in hydrophobic interactions between multiple proline and tyrosine residues on the border of the monomer boundary in the homodimer. Monomers A/D or B/C interact through the symmetric peripheral 173 flap, 97 and 60 loops. Both 97 loops on each segment experience Van der Waals forces between segments 95-99 due to their close proximity between monomers, with the Ile 99 side chains directly touching each-other. . There is additional hydrophobic interaction at the outer edge of the A/D or B/C monomer pairs between Pro 60A-Asp 60B and Gly 173B and Tyr 173D. They are also cross-linked via a salt bridge between Asp 60B and Arg224; as well as through several hydrogen bonds artifically calculated below.
In order for active beta tryptase tetramers to form, a heparin polysaccharide is required to bind to the enzyme. It forms a bridge between the two homodimers, stabilizing the complex as a whole, meaning that the aforementioned macromolecule’s dissociation causes the enzyme to break into inactive monomers. Acidity is an important factor for tetramer formation; with active beta tryptase enzymes only forming at pH 6 or below — the crystal used in this paper was formed at pH 5. This is due to the importance of histidine residues (and specifically the imidazole side chains) in the binding of heparin, with His 106 and His 108 as the two main actors in this process. At pH 7 and above, imidazole is neutrally charged, while acidic conditions make it positively charged, enabling binding with the highly negatively charged heparin ligand. Recent studies have highlighted the possibility of DNA-stabilized beta tryptase, given the former molecule’s highly electronegative backbone. Although this likely has few uses in vivo, given the granule release mechanism and perpetrating role of beta tryptase in inflammation, it does highlight that heparin’s stabilization role is theoretically not unique to the molecule. The heparin sulfate groups also interact via salt-bridge with separate positively charged regions on the periphery of each homodimer, which contain groups of lysine, argenine, and histidine residues.
Scientists use blood serum levels of beta tryptase and similar proteases as a bioindicator of asthmatic or anaphylactic reactions, given their uniquely distinctive roles as irritants in both reactions. However, not all mast cells cause the same levels of inflammation. In healthy patients, the majority of airway mast cells contain exclusively tryptase within the granules, while asthmatic patients are more likely to have a higher proportion of the aforementioned cell type with tryptase, chymase, and carboxypeptidase A3 protease proteins. This complicates the scientific narrative, since beta tryptase’s cleavage of PAR2 is associated with the initiation of the misguided respiratory tract immune response, yet unto itself is not sufficient to trigger the asthmatic or anaphylactic phenotype.
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