The accumulation of β-amyloid proteins (Aβ) in the brains of persons with Alzheimer’s disease is considered a hallmark of the disease’s pathology. The tetrameric enzyme γ-secretase performs the ultimate cleavage of amyloid precursor protein (APP) to produce the pathogenic Aβ proteins. The two most common Aβ products are Aβ40 and Aβ42. Differing only by two residues at the C-terminus, Aβ42 is significantly more hydrophobic and subsequently has an increased aggregation capacity. As such, Aβ42 is considered more pathologically relevant, and an increased ratio of AB42:AB40 has been shown to lead to increased Aβ plaque deposition (1). The catalytic component of γ-secretase, presenilin (PS), exists in two homologues – PS1 and PS2 – that both form active enzymes and have been shown to produce different amounts of Aβ40 and Aβ42, leading to differing Aβ42:Aβ40 generation ratios. While small molecules have been identified that are capable of shifting the Aβ42:Aβ40 ratio (γ-secretase modulators) (2), their discovery and optimisation has been hampered by a limited understanding of the structure and dynamics of γ-secretase, as well as a limited understanding of how the PS isoforms influence product generation by γ-secretase. This study examined the Aβ generation pathways using metadynamics to identify low energy states of PS1- and PS2-containing γ-secretases bound to the various Aβ substrates. Our work shows that PS1-containing γ-secretase exhibits a broader conformational ensemble when bound to the initial substrate of the Aβ40 processing pathway, while the PS2-containing complex exhibits a broader conformational ensemble when bound to the initial substrate of the Aβ42 processing pathway. These results suggest that PS1-containing enzymes may yield greater Aβ40, while PS2-containing enzymes may yield greater Aβ42, recapitulating observed product distributions in cell-based studies. Our results further provide a pathway for the structure-based rational design of γ-secretase modulators.