A genetic selection elucidates structural determinants of arginine versus lysine specificity in trypsin

Gene. 1993 Dec 27;137(1):121-6. doi: 10.1016/0378-1119(93)90259-6.


A genetic selection has been used to isolate variants of the serine protease, trypsin (Tsn), altered in specificity toward lysine- and arginine-containing substrates. Growth of a lysine auxotroph of Escherichia coli was coupled to activation by Tsn of a non-nutritive source of lysine present in selective media. Nine Tsn variants possessing partial activities were isolated from a random library encompassing amino acids 189 and 190 at the base of the primary specificity pocket. Functional analysis of these isolates indicates that preservation of activity toward lysine-containing substrates is more tolerant to mutation than is activity toward equivalent arginine-containing substrates. Both the position, as well as the accessibility to substrate, of a negatively charged group in the binding pocket appear critical to maintenance of high-level catalytic potency by Tsn.

MeSH terms

  • Arginine / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Genetic Variation
  • Lysine / metabolism*
  • Protein Engineering / methods*
  • Substrate Specificity*
  • Trypsin / metabolism*


  • Arginine
  • Trypsin
  • Lysine