Definition of the extended substrate specificity determinants for beta-tryptases I and II.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.

Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme.

Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin-like serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two-step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post-translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.

Factors that modify the molecular size of phospholamban, the 23,000-dalton cardiac sarcoplasmic reticulum phosphoprotein.

Phospholamban, a 23,000-dalton phosphoprotein present in cardiac muscle sarcoplasmic reticulum vesicles is quantitatively dissociated into three smaller sized phosphorylated components of 11,000, 15,000, and 20,000 daltons when sarcoplasmic reticulum, solubilized in 1% (w/v) sodium dodecyl sulfate and 1 mM MgCl2, is heated at 71 degrees C or greater for 2 min. This dissociation is inhibited by Mg2+ (50% at approximately 5 mM). The 23,000-dalton phosphoprotein reformed from the 11,000-, 15,000-, and 20,000-dalton phosphorylated components when phosphorylated sarcoplasmic reticulum that had been boiled in 1% sodium dodecyl sulfate and 1 MM MgCl2 was stored for 1 week at -70 degrees C. We propose that the 23,000-dalton phosphorylated protein is a trimer, composed of three subunits with molecular mass of 11,000, 8,000, and 4,000 daltons. In this model, only the 11,000-dalton subunit would be phosphorylated. The partial dissociation of the 23,000-dalton phosphorylated protein would result in the formation of the 19,000 (11,000 + 8,000)-dalton or the 15,000 (11,000 + 4,000)- dalton phosphorylated components. Full dissociation of the 23,000-dalton phosphorylated protein would result in the formation of the 11,000-dalton phosphorylated component.