FORMATION AND ACTIVITIES OF SUBSTITUTED PHENACYLCHYMOTRYPSINS

1. 1.|The preparation of derivatives of α-chymotrypsin with p-nitrophenacyl bromide, phenacyl bromide and p-methoxyphenacyl bromide is described. 2. 2.|For the p-nitrophenacyl derivative, the new 350-nm absorption is pH independent from pH 2.88 to 11.25, mitigating against a major change in environment at the new chromophore over this range. 3. 3.|The enzyme derivatives (likely at Met-192) show different decreased but definite catalytic activities towards substrates of α-chymotrypsin. In each case, this activity is due to the modified enzyme itself. 4. 4.|Phenacyl- and p-methoxyphenacylchymotrypsin show generally increased dissociation constants and decreased catalytic constants. Below pH 8.5, p-nitrophenacylchymotrypsin towards ethyl-N-acetyl-l-tryptophanate has only decreased kcat values compared to native enzyme, Km being unaltered. The pH dependencies of kcat below pH 8.5 and Km over the entire range are identical for parent and nitrophenacyl enzyms. Above pH 8.5, kcat for the enzyme derivative increases and is still rising at pH 10, having reached over twice its value at pH 8.5. The effect is reversible. 5. 5.|These results are taken to indicate that the conformational change occurring in α-chymotrypsin at pK 9 occurs for the nitrophenacyl enzyme as well, but, in the latter case, it reverses the original inhibition of kcat by the nitrophenacyl group. © 1969.