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UBC Theses and Dissertations

Bioengineering coagulation factor Xa substrate specificity into Streptomyces griseus trypsin Page, Michael J.


Extended substrate specificity is exhibited by a number of highly evolved members of the SI peptidase family, such as the vertebrate blood coagulation proteases. Dissection of this substrate specificity has been hindered by the complexity and physiological requirements of these proteases. In order to understand the mechanisms of extended substrate specificity, a bacterial trypsin-like enzyme, Streptomyces griseus trypsin (SGT), was chosen as a scaffold for the introduction of extended substrate specificity through structure-based genetic engineering. Recombinant and mutant SGT proteases were produced in a B. subtilis expression system, which constitutively secretes active protease into the extracellular medium at greater than 15 mg/L of culture. Comparison of the recombinant wild-type protease to the natively produced enzyme demonstrated near identity in enzymatic and structural properties. To begin construction of a high specificity protease, four mutants in the S1 substrate binding pocket (T190A, T190S, T190V, and T190P) were produced and examined for differences in the Arg:Lys preference. Only the T190P mutant of SGT demonstrated a significant increase in P1 arginine to lysine preference - a three-fold improvement to 16:1 - with only a minor reduction in catalytic activity (k[sub cat] reduction of 25%). The 1.9 Å resolution crystal structure of T190P mutant of SGT in complex with the small molecule inhibitor benzamidine was subsequently determined. The model shows that the increased preference for Arg over Lys side chains in the S1 pocket is the result of the second shell residues of the S1 pocket, particularly by the N-terminal residue of the protease which does not conflict with the introduced proline ring. Using the T190P mutant of SGT as a starting point, coagulation factor Xa (FXa) substrate specificity determinants were then introduced by additional site-directed mutagenesis. To aid in purification of the recombinant proteases a hexa-histidine tag was added to the C-terminus of the protein. Addition of the purification tag reduced the ability of the expression host to produce the enzyme (3 mg/L of culture) but simplified the purification of SGT from the culture medium. Various combinations of a two-residue loop and a number of point mutations at positions 99, 172, 174, 180, and 217 were constructed and characterized in SGT. The mutant bearing mutations at all positions except residue 217 demonstrated a moderate preference for FXa substrates as determined using chromogenic synthetic peptides. However, the kinetic properties of the mutant enzyme suggested that the 172-loop, a member of the S3/S4 substrate binding pocket, is not in a conformation similar to FXa. Addition of the Y217E mutation was designed to stabilize the loop but led to a specific protease similar to coagulation factor XIa and not FXa. These results confirm the evolutionary relationship amongst the vertebrate coagulation proteases and demonstrate the importance and flexibility of the 172-loop. Further, Na⁺ binding, a novel property found in several coagulation proteases, is suggested to play a role in stabilization of the 172-loop and in turn played an important role in the evolution of the vertebrate coagulation cascade.

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