Kinetic mechanism of ENPP1 ATPase: Implications for aberrant calcification disorders and enzyme replacement therapy.

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is a transmembrane glycoprotein enzyme with an extracellular catalytic domain that hydrolyzes ATP into AMP and pyrophosphate (PPi). The ENPP1 ATPase is the major source of extracellular PPi, a critical physiological regulator of calcium phosphate crystal formation and biomineralization. ENPP1 deficiency lowers systemic PPi levels and induces life-threatening arterial calcifications. Enzyme replacement therapy with a soluble ENPP1 biologic restores plasma PPi and eliminates calcification and associated mortality in ongoing clinical trials in patients with ENPP1 deficiency. Despite the significant role of ENPP1 in inhibiting vascular calcification and regulating mammalian biomineralization via extracellular PPi levels, little is known about the molecular mechanism of PPi liberation by ENPP1. Here, we provide a kinetic analysis of the ENPP1 catalytic ATPase cycle. Our analysis shows that ATP cleavage, PPi release, and hydrolysis of the covalent AMP-ENPP1 intermediate are rapid (>1000 s-1) and that AMP product release is slow and rate-limiting. The steady-state Michaelis constant of ATP substrate (KM,T) is comparable to physiological serum ATP levels of ∼100 nM, rendering ENPP1 activity sensitive to small changes in serum ATP. AMP binds strongly, with an affinity comparable to KM,T, such that ENPP1 is subject to intrinsic regulatory feedback by AMP under physiological concentrations of ∼100 nM. This product inhibition can attenuate ENPP1 during periods of high PPi liberation, maintaining relatively constant plasma PPi levels. The quantitative parameters of the ENPP1 ATPase cycle provided here allow for predictable outcomes of ENPP1 enzyme replacement therapy and provide plausible expectations for other PPi-linked calcification disorders.