Kinetics of action of pepsin on fluorescent peptide substrates

摘要: Oligopeptide substrates of porcine pepsin (E) the type A-Phe-Phe-B (S) that are cleaved solely at Phe-Phe bond under conditions these studies, and bearing an amino-terminal fluorescent probe group (mansyl or dansyl), have been used for stopped-flow measurements rate formation A-Phe product. These experiments were conducted [E] greater than [S], kinetic data compared with those obtained [S] Phe-B product (the same in all cases). The results A = mansyl-Gly, mansyl-Gly-Gly, dansyl-Gly-Gly support conclusion rate-limiting step over-all catalytic process is associated scission first detectables ES complex. Although this varies widely nature portion A-Phe-Phe-B, magnitude dissociation constant relatively invariant. This supports view that, cleavage oligopeptide by pepsin, secondary enzyme--substrate interactions may cause conformational changes site, a total binding energy be attainment transition state bond-breaking step. With hydrolyzed extremely rapidly (A dansyl-Gly-Ala, dansyl-Ala-Ala), appears to faster steady-state rate, suggesting additional has become kinetically significant process. return conformation active site its original state.