1 research outputs found
Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592
The sterile alpha
motif (SAM) and histidine-aspartate (HD) domain
containing protein 1 (SAMHD1) constitute a triphosphohydrolase that
converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides
and triphosphates. SAMHD1 exists in multiple states. The monomer and
apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP
at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site
1 (AS1), induces formation of the tetramer, the catalytically active
form. We have developed an enzyme kinetic assay, tailored to control
specific dNTP binding at each site, allowing us to determine the kinetic
binding parameters of individual dNTPs at both the AS2 and catalytic
sites for all possible combinations of dNTP binding at both sites.
Here, we show that the apparent <i>K</i><sub>m</sub> values
of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP <
dTTP. Interestingly, dCTP binding at AS2 significantly reduces the
dCTP hydrolysis rate, which is restored to a rate comparable to that
of other dNTPs upon dGTP, dATP, or dTTP binding at AS2. Strikingly,
a phosphomimetic mutant, Thr592Asp SAMHD1 as well as phospho-Thr592,
show a significantly altered substrate specificity, with the rate
of dCTP hydrolysis being selectively reduced regardless of which dNTP
binds at AS2. Furthermore, cyclin A2 binding at the C-terminus of
SAMHD1 induces the disassembly of the SAMHD1 tetramer, suggesting
an additional layer of SAMHD1 activity modulation by cyclin A2/CDK2
kinase. Together, our results reveal multiple allosteric mechanisms
for controlling the rate of dNTP destruction by SAMHD1