The human neuroendocrine enzyme glutamic acid decarboxylase (GAD) catalyses the synthesis of the neurotransmitter GABA, using pyridoxal-5’-phosphate (PLP) as cofactor. GAD exists as two isoforms named according to their respective molecular weights, GAD65 and GAD67. While cytosolic GAD67 is more saturated with the co-factor PLP and constitutively active to produce basal levels of GABA, the membrane associated GAD65 exists mainly as autoinactivated apoenzyme. ApoGAD65 can be reactivated by PLP to form holoGAD65 when additional GABA is required, for example in response to stress. GAD65, but not GAD67, is a major autoantigen and autoantibodies to GAD65 are detected at high frequency in patients with type 1 diabetes and other autoimmune disorders. Although GAD65 autoinactivation appears to be linked to its autoantigenicity, neither are understood in any molecular detail. We have used both computational and experimental methods in order to characterise the conformational nature of holo- to apo- conversion in GAD65, and thus its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain and catalytic loop that drives opening and autoinactivation, consistent with proteolysis fragmentation patterns. We present a model of inactive apoGAD65 in an open conformation that is consistent with fluorescence spectroscopy and SAXS data. Our studies provide an overall description of conformational opening of GAD65, and the dynamic communication between domains that drive this process. Understanding GAD65 as a conformational ensemble rather than a static structure may alter considerably the interpretation of two decades of epitope mapping data, and may have consequences for other enzyme autoantigens.