Spermine is an endogenous small‐molecule 20S proteasome activator synergistically coupling chymotrypsin‐like active‐site modulation with α‐ring gate opening

The biogenic polyamine spermine accumulates to millimolar concentrations in proliferating cells, yet its direct effect on the 20S proteasome ( EC3.4.25.1 ) has remained mechanistically obscure. Here, we show that spermine directly binds and activates the human 20S proteasome through two mechanistically distinct, kinetically separable events. At low millimolar concentrations, spermine selectively stimulates the chymotrypsin‐like β5 site up to 14‐fold over the basal rate, whereas above ~1.7 m

it triggers a highly cooperative (Hill n  = 8) opening of the α‐ring channel that additionally engages the trypsin‐ and caspase‐like sites. Simultaneous occupancy of both regions generates a hybrid enzyme form for which the catalytic constant is super‐additive, 163‐fold over k _cat . The α‐ring transition is intrinsically slow, with channels reaching only 44% of equilibrium opening within the first 5 min. Abolition of this hysteresis by SDS or by PA28αβ preloading confirms that gate opening is the rate‐limiting step. The combination of a high cooperative threshold ( K _ch  ~ 1.7 m ) and slow gating kinetics defines a kinetic filter that allows the proteasome to respond selectively to sustained elevations of free polyamines, such as those characteristic of proliferating and malignant cells, while remaining insensitive to transient metabolic fluctuations. Finally, we show that spermine enhances hydrolysis of unfolded substrates by the 20S proteasome in vitro and in mammalian cells. Spermine thus emerges as the first endogenous small‐molecule activator that engages both the catalytic and gating layers of 20S regulation, linking polyamine metabolism directly to proteolytic capacity.