Abigail Williams | 2026 I.S. Symposium
狈补尘别:听Abigail Williams
罢颈迟濒别:听Investigating Stichopus japonicus Arginine Kinase (SjAK) to Understand the Evolution of Negative Cooperativity in Phosphagen Kinases
Major: Biochemistry and Molecular Biology
础诲惫颈蝉辞谤蝉:听Dean Fraga; Mark Snider
Complex biochemical traits arise through protein evolution, allowing proteins to take on diverse roles in a biological system. Cooperativity, when substrate binding of one subunit in a multimeric protein changes substrate affinity of the other, is an example of a complex trait that has evolved in some proteins within the phosphagen kinase (PK) family. Structural analysis reveals that Stichopus japonicus arginine kinase (SjAK), a dimeric, cooperative PK, as well as other cooperative PKs, contains an internal hydrogen bond network. The objective of this study was to investigate the functional and evolutionary significance of residues within this network in SjAK. S194, a residue that plays a central role in this network, was substituted with alanine and threonine. SjAK WT, S194A, and S194T were purified and biochemically characterized via size exclusion chromatography, UV-Vis linked kinetic assays, and ITC cooperativity assays to determine the quaternary structure, kinetic parameters, and degree of cooperativity, respectively. The catalytic turnover of both variants was 3-fold lower than WT, and the KM of S194T was 3-fold greater than that of WT, suggesting that S194T may have reduced arginine affinity. ITC cooperativity assays were inconclusive, but structural analysis revealed that S194T may not undergo the same conformational changes as WT upon ATP binding. Because ATP binding has a synergistic effect in PKs, this could explain why S194T has a greater KM value. Overall, these findings show how seemingly neutral residues located outside of the active site or dimer interface can have an effect on protein structure and enzymatic activity.
Posted in Symposium 2026 on May 1, 2026.
