The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity

Citation
A. Pasternak et al., The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity, PROTEIN SCI, 10(7), 2001, pp. 1331-1342
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
7
Year of publication
2001
Pages
1331 - 1342
Database
ISI
SICI code
0961-8368(200107)10:7<1331:TECOIF>2.0.ZU;2-E
Abstract
The contribution of induced fit to enzyme specificity has been much debated , although with little experimental data. Here we probe the effect of induc ed fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations ar e observed between BPTI affinity and the values of k(cat)/K-m for the hydro lysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, Delta I16V17/D194N, and Delta I16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, Delta I16V17 trypsinogen, is t he lone outlier in the correlations; its activity is lower than expected ba sed on its affinity for BPTI. The S1 site and oxyanion hole, formed by segm ents 184A-194 and 216-223, are trypsinlike in all of the enzymes. These str uctural and kinetic data confirm that BPTI induces an active conformation i n the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor change s in the energetic cost of inducing a trypsinlike conformation, Although th e S1 site and oxyanion hole are similar in all four variants, the N-termina l and autolysis loop (residues 142-152) segments have different interaction s for each variant. These results indicate that zymogen activity is control ed by a simple conformational equilibrium between active and inactive confo rmations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not gene rally contribute to enzyme specificity.