Understanding the allosteric regulation of Geobacillus stearothermophilus L-lactate dehydrogenase (GsLDH), a homotetrameric enzyme, is crucial for its application in industrial biocatalysis due to its role in lactate-pyruvate interconversion and NAD+/NADH cofactor regeneration. In this study, we combined experimental and computational approaches to elucidate the mechanisms underlying the enzyme’s allosteric activation by fructose 1,6- bisphosphate (FBP), which stabilizes tetramerization and enhances substrate affinity for pyruvate. We compared the wild-type (wt) enzyme with a triple mutant and a single mutant to assess the contributions of oligomerization and allosteric modulation. Our findings reveal that the triple mutant retains its tetrameric structure and high substrate affinity regardless of FBP. In contrast, the single mutant maintains tetramerization but lacks the enhanced substrate affinity, suggesting that oligomerization alone is insufficient to propagate the allosteric signal. Both oligomerization and allosteric modulator binding are necessary to establish communication pathways that effectively modulate substrate affinity, as demonstrated by significant changes in Km. To investigate dynamic conformational changes, we employed molecular dynamics (MD) simulations and applied our newly developed tool, MDavocado. This computational tool visualizes MD trajectories using time-resolved Ramachandran plots, effectively aggregating millions of data points into interpretable visualizations. This approach enables rapid identification of flexible regions and global motion patterns across all amino acids. The application of MDavocado highlighted how FBP-induced oligomerization correlates with coordinated backbone dynamics, reinforcing the necessity of dual structural and allosteric inputs for optimal enzyme function. Our findings advance the understanding of structure- allostery relationships in industrially relevant enzymes and underscore the utility of MDavocado in resolving complex protein dynamics.