UBE3A is a HECT (homologous to E6AP C-terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin-proteasome pathway. The UBE3Agene is of unique interest for its gene dosage-dependent effect in the developing brain: Precise deletion or null mutation of the maternal copy of UBE3A causes a severe intellectual disability known as Angelman syndrome; meanwhile, duplication or triplication of the gene region in which UBE3A resides is linked to a prevalent syndromic form of autism known as Dup15q syndrome. However, little is known about the effects of missense variants which cause a single amino acid change in the enzyme, and prediction of disease outcomes for a given variant remains a challenge. Here, we pose that investigating variants’ effects on UBE3A functional activity levels is critical for predicting disease. In order to identify if precise mutations in UBE3A are sufficient to drive disease, we devised a high-throughput assay to screen the functional consequence of UBE3A missense variants. We screened over 150 variants and identified distinct functional classes of UBE3A mutants based on their effect on enzymatic activity. Importantly, we identified over a dozen novel gain-of-functionvariants that aberrantly hyperactivate UBE3A enzyme activity. Through collaborations with clinical centers, we confirm that individuals possessing hyperactivating UBE3A variants exhibited phenotypes that were distinguishable from Angelman. Mice carrying a specific hyperactivating mutation on the maternal allele exhibited aberrant motor and early communication defects, as well as microcephaly. Finally, we mapped the results of our screen to the UBE3A protein structure to reveal a previously-undefined allosteric regulatory exosite within the catalytic domain that we show to act as a charge-dependent regulator of enzymatic activity. We found additional HECT domain enzymes to possess disease-associated variants within their exosites, suggesting that exosite dysfunction is a common mechanism underlying a set of neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that deep structure-functional analysis of protein variants can uncover disease-relevant regulatory mechanisms.