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Conversion of trypsin to a functional threonine protease

¹ University of California, San Francisco, Department of Pharmaceutical Chemistry, San Francisco, California 94143-2280, USA
² San Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, USA

(RECEIVED February 21, 2006; FINAL REVISION March 7, 2006; ACCEPTED March 8, 2006)

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the -carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10²–10⁴-fold less active than wild type in k_cat/K_M but >10⁶-fold more active than the Ser 195 Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'– P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.

Keywords: serine proteases; trypsin; enzyme mechanisms; protein engineering; substrate specificity

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