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Gene. 1993 Dec 27;137(1):121-6.

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

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Department of Pharmaceutical Chemistry, University of California San Francisco 94143-0446.


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.

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