The influence of Zn2+-binding motif sequence on the catalytic activity of transthyretin towards amyloid beta

Abstract

The proteolysis property of transthyretin (TTR) was believed to be important for physiological processes and have therapeutic potential. In the present study, we interchanged the corresponding sequences of Zn 2+ binding motif between human and Crocodylus porosus TTRs. The kinetic parameters of the proteolytic activity towards amyloid beta 1-42 (Aβ 1-42 ) of the mutated TTRs were determined by using a sensitive fluorescence-based method, and compared with the wild types. The results showed that C. porosus TTR with Zn 2+ -binding motif sequence of human TTR (hu-motif/crocTTR) has apparent K m (K app m ) of 118 ± 13 µM which was significantly higher than those for human TTR (53.6 ± 1.5 µM), C. porosus TTR (crocTTR) (30.2 ± 4.0 µM), and human TTR with Zn 2+ -binding motif sequence of C. porosus TTR (croc-motif/huTTR) (53.4 ± 0.87 µM). In addition, the catalytic efficiency (V max /K app m ) of croc-motif/huTTR (1.16 ± 0.0066 RFU min −1 µM −1 ) was significantly higher than human TTR (0.639 ± 0.0085 RFU min −1 µM −1 ) but similar to those of crocTTR (1.11 ± 0.082 M −1 min −1 ) and hu-motif/crocTTR (0.894 ± 0.031 RFU min −1 µM −1 ). The obtained results provided insight of the influence of the Zn 2+ -binding motif sequence and the evolutionary change structure on the proteolytic activity towards Aβ of TTR and useful information for designing a therapeutic TTR.