A GENETIC SELECTION ELUCIDATES STRUCTURAL DETERMINANTS OF ARGININE VERSUS LYSINE SPECIFICITY IN TRYPSIN

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