REASSESSMENT OF THE CATALYTIC ACTIVITY AND SUBSTRATE SPECIFICITY OF FKBP35 FROM Plasmodium knowlesi USING PROTEASE-FREE ASSAY Cahyo Budiman 1* , Carlmond Goh Kah Wun 1 , Lee Ping Chin 2 , Rafda Razali 1 , Thean Chor Leow 3 1 Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia 2 Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia 3 Enzyme and Microbial Technology Research Center, Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia *Corresponding author’s email: cahyo@ums.edu.my Received date: 1 September 2020 | Accepted date: 21 September 2020 ABSTRACT FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confrm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specifcity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with k cat /K M of 4.5 + 0.13 × 10 5 M −1 s −1 , while the k cat /K M of active site mutant of D55A is 0.81 + 0.05 × 10 5 M −1 s −1 . These values were considered comparable to k cat /K M obtained from the protease-coupling assay. Interestingly, the substrate specifcities Borneo International Journal of Biotechnology (BIJB) Vol. 1 (December 2020), 125 – 143 e-ISSN 2716-697X