view article
Figure 3
In vitro validation of the inhibitory mechanism. (a) Activity (%) of astacin (at 1 nM concentration), meprin β (at 1 nM concentration) and ovastacin (at 5.6 nM) in the presence of fetuin-B variants at 300 nM (CPDC and CPRC) or 12.5 nM (other variants). Full astacin, meprin β and ovastacin activities (100%) correspond to substrate-turnover rates of 35.4 ± 5.7, 7.7 ± 0.2 and 10.7 ± 0.8 nM s−1, respectively; w/o, without fetuin; hFB, human fetuin-B; bFB, bovine fetuin-B; mFB, murine fetuin-B; mFBCY2, CY2 domain of mFB; mFBCTR, CTR domain of mFB; mFBC39S,C374S, double point mutant of murine fetuin-B; CPDC and CPRC, cyclic peptides comprising the respective sequences. Experiments were performed in triplicate; error bars indicate standard deviations. (b) Plot of fractional velocity (logarithmic scale) with different fetuin-B variants: mFB, murine fetuin-B; mFBABA, variant with CY1 and CTR from mouse fetuin-A and CY2 from mFB; mFBH1-swap, mutant replacing CY2 hairpin I with CY1 hairpin I; mFBP155A, mFBD156A, mFBP155A,D156A and mFBC154S,C157S are point mutants of murine fetuin-B. Enzyme and substrate concentrations were 1.0 nM and 195 µM for astacin, 0.5 nM and 26 µM for meprin β and 0.7 nM and 26 µM for ovastacin, respectively. Error bars indicate standard deviations. (c) Ki values determined from (b) using Morrison's equation with the respective standard errors (SE).

IUCrJ
Volume 6| Part 2| March 2019| Pages 317-330
ISSN: 2052-2525
https://doi.org/10.1107/S2052252519001568