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 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.
Perona
J J
JJ
Department of Pharmaceutical Chemistry, University of California San Francisco 94143-0446.
Evnin
L B
LB
Craik
C S
CS
D001120:Arginine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D008239:Lysine / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
D001120:Arginine; D014357:Trypsin; D008239:Lysine