Synergistic Effects of Distal Mutations on Enzyme Activity: A Comprehensive Study

The distal mutation is likely to exert a discernible impact on the enzyme activity within the range of 58-62. Recognizing this, F308G was employed as a template to effectuate further mutations on F274, incorporating H, K, R, and L. Notably, F274H, F274K, and F274R mutants exhibited a synergistic effect with F308G (as depicted in Figure 4F). Subsequently, the mutant N96S/A271E/F308G was utilized as a template to introduce mutations on F274, with Y being inserted at position 274 owing to its presence in the pSUFER space and possession of a similar side chain as F. The results indicated that the activity of all four quadruple mutants was augmented, with the N96S/A271E/F274Y/F308G mutant displaying a staggering 56-fold increase in activity (as illustrated in Figure 4F).