Abstract

Cytosolic creatine kinases (CK) play a central role in buffering ATP levels and are obligate dimers; a single subunit cannot function without the presence of the other. However, a recent discovery revealed that novel CKs in Perkinsus marinus (PmCK), a parasitic protozoan, comprise only a single subunit yet remain completely active. Amino acid sequence alignment indicated that PmCKs are missing some key residues involved in dimerization as identified in dimeric CKs; hence they exist as monomers. However, the monomeric state of PmCK is evolutionarily in query: whether it lost the ability to dimerize or is only a few mutations away from becoming a dimer. To understand how the change in key residues affects the quaternary structure of PmCK, site-directed mutagenesis was used to introduce the key residues (G83D, F178R, and G179D) into PmCK. Five mutants were made: G83D, F178R, D179G, G83D/F178R (double), and G83D/F178R/D179G (triple). Size-exclusion chromatography showed that the Double and Triple mutants formed equilibrium mixtures of monomer and dimer (~45 and ~90 kDa, respectively), with the monomeric form being predominant. NADH-coupled enzyme assay indicated that both mutants showed significant decreases in catalytic efficiency (~20 and 65-fold decrease, compared to that of the WT). It is uncertain why the dimers showed decrease in activity; hence it is difficult to support or refute whether PmCK might have escaped from its dimeric precursor or never acquired the additional ‘background/hidden’ mutation needed to become a functional dimer.

Advisor

Fraga, Dean

Department

Biochemistry and Molecular Biology

Disciplines

Biochemistry | Molecular Biology

Keywords

phosphagen kinase, creatine kinase, evolution, dimerization, Perkinsus marinus, quaternary structure, site-directed mutagenesis

Publication Date

2023

Degree Granted

Bachelor of Arts

Document Type

Senior Independent Study Thesis

Download

Share

COinS
 

© Copyright 2023 Kyungjoo Kim